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Investigating the molecular architecture of yeast histone acetyltransferase complexes Setiaputra, Dheva 2016

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  INVESTIGATING THE MOLECULAR ARCHITECTURE OF YEAST HISTONE ACETYLTRANSFERASE COMPLEXES  by  DHEVA SETIAPUTRA B.Sc. The University of British Columbia, 2011   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  December 2016 ©Dheva Setiaputra 2016 ii  Abstract  Post-translational modification of histones, such as the addition of acetyl groups, is a major regulatory mechanism for gene expression. Histone acetylation is catalyzed by highly conserved lysine acetyltransferase (KAT) enzymes that are often part of large, modular, and multifunctional complexes. Despite their fundamental importance, the reasons behind the tendency of these enzymes to form large complexes remain unclear. We investigated the organization of these complexes by elucidating the molecular architecture of three yeast KAT complexes: Spt-Ada-Gcn5 Acetyltransferase (SAGA), nucleosomal acetyltransferase of histone H4 (NuA4), and Elongator. The yeast SAGA complex is the largest KAT complex in yeast, and activates the expression of many stress response genes. Mutations of its human homologues have been implicated in spinocerebellar ataxia and oncogenesis. Using single particle electron microscopy and crosslinking coupled to mass spectrometry, we show that the catalytic module of SAGA resides within a highly flexible tail adjacent to numerous chromatin-binding subunits. We propose that the flexible SAGA tail is the nucleosome-interacting surface, and its plasticity serves to accommodate the various configurations of the chromatin substrate. NuA4 is another KAT complex whose catalytic subunit, Esa1, is the only essential KAT in yeast. NuA4 has highly conserved roles in the expression of housekeeping genes and the DNA damage repair pathway. Its subunits organize into modules that act independently of the complex. We show that these moonlighting modules form distinct globular structures that are peripherally associated with NuA4, which likely facilitates their dynamic nature. Similar to iii  SAGA, NuA4 subunits that bind chromatin surrounds its catalytic subunit, possibly positioning its substrate nucleosome for efficient acetylation. Yeast Elongator, consisting of two copies each of six unique subunits, was initially characterized as a component of the elongating RNA polymerase II holoenzyme with histone acetyltransferase activity. However, further research has revealed a prominent role for the complex in modifying the wobble base pair of tRNAs. We generated the first three-dimensional reconstruction of Elongator and show that it organizes asymmetrically, with the two copies of its catalytic subunit residing in very different environments. Our structural investigations represent the first steps towards understanding the molecular mechanisms of these enigmatic complexes.            iv  Preface Chapter 2 of this dissertation is a version of a study published in whole in the Journal of Biological Chemistry (See below). As the first author of this publication, I designed all the experiments, optimized the purification of the complex, and conducted all of the microscopy and data processing. James Ross assisted in strain preparation and purification of the GFP-tagged complex. Derrick Cheng contributed to the transcriptional binding activator experiments. Shan Lu and Meng-Qiu Dong conducted the crosslinking coupled to mass spectrometry experiment and analysis. I was responsible for writing the manuscript and preparing all of the figures, with extensive assistance from Calvin Yip. Note that this manuscript refers to lysine acetyltransferases (KATs) using the slightly more outdated term histone acetyltransferases (HATs). This chapter retains the usage of this term to be more congruous with the published article. Sections 2.2.12, 2.3.10, and 2.4.3 were not part of the publication. Setiaputra D., Ross J.D., Lu S, Cheng D.T.H., Dong M.Q., and Yip C.K. Conformational Flexibility and Subunit Arrangement of the Modular Yeast Spt-Ada-Gcn5 Acetyltransferase Complex. J. Biol. Chem. 290: 10057-10070. (2015).  Chapter 3 of this dissertation is a version of a manuscript in preparation with contributions from James Ross, Shan Lu, Meng-Qiu Dong, and Jacques Côté. I optimized the purification and conducted all of the microscopy and data processing for both NuA4 and TIP60. James Ross prepared the GFP-tagged strains and participated in initial purifications of NuA4. Shan Lu and Meng-Qiu Dong conducted the crosslinking coupled to mass spectrometry experiments and data analysis. Jacques Côté provided purified human TIP60 complex. I was responsible for writing the manuscript and preparing the figures. Chapter 4 of this dissertation is a version of a manuscript for EMBO Reports (see below). As the first author of this publication, I designed all of the experiments, conducted the v  purification in alternate salt buffers and the Elongator containing two Elp456 subcomplexes, and conducted most of the microscopy and data processing. Derrick Cheng optimized the purification of the complex and generated the strains for subunit localization of the complex. Jesse Hansen conducted large scale purification of the complex for the three dimensional reconstruction. Udit Dalwadi prepared the recombinant Elp456 subcomplex and conducted some of the electron microscopy image acquisition and data processing. Jeffrey To and Cindy Lam were responsible for the phenotypic analysis of Elp2 mutants (Section 4.3.3), including strain and plasmid construction as well as phenotyping. Shan Lu and Meng-Qiu Dong conducted the crosslinking coupled to mass spectrometry experiments and data analysis. I was responsible for writing the manuscript and preparing the figures, with extensive assistance from Calvin Yip. Setiaputra D., Cheng D.T.H., Lu S., Hansen J.M., Dalwadi U., Lam C.H.Y., To J.L., Dong M.Q., and Yip C.K. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement. EMBO R. e201642548 (2016).             vi  Table of contents Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iv Table of contents ............................................................................................................................ vi List of tables .................................................................................................................................. xii List of figures ............................................................................................................................... xiii List of abbreviations ..................................................................................................................... xv Acknowledgments...................................................................................................................... xviii Chapter 1: Introduction ................................................................................................................... 1 1.1 Organization of eukaryotic chromatin................................................................................... 2 1.2 Transcription and chromatin in yeast .................................................................................... 4 1.3 Histone post-translational modifications ............................................................................... 5 1.3.1 Histone acetylation ......................................................................................................... 5 1.3.2 Histone methylation ........................................................................................................ 6 1.3.3 Histone ubiquitination .................................................................................................... 8 1.4 Protein domains found in lysine acetyltransferases .............................................................. 9 1.4.1 Histone acetyltransferase domains ................................................................................. 9 1.4.2 Ubp8 histone deubiquitinase domain ........................................................................... 16 1.4.3 Chromatin readers ......................................................................................................... 17 1.5 Lysine acetyltransferase complexes .................................................................................... 19 vii  1.5.1 Spt-Ada-Gcn5 Acetyltransferase complex (SAGA) ..................................................... 20 1.5.2 Nucleosomal Acetyltransferase of histone H4 (NuA4) ................................................ 22 1.5.3 Elongator ...................................................................................................................... 24 1.5.4 Mammalian KAT complexes ........................................................................................ 26 1.6 Techniques for analyzing KAT complexes ......................................................................... 28 1.7 Objectives of thesis ............................................................................................................. 30 Chapter 2: Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 Acetyltransferase complex .................................................................................................. 32 2.1 Introduction ......................................................................................................................... 32 2.2 Experimental methods ......................................................................................................... 34 2.2.1 S. cerevisiae strain construction ................................................................................... 34 2.2.2 Purification of native SAGA ........................................................................................ 34 2.2.3 Negative stain sample preparation and imaging ........................................................... 37 2.2.4 Image processing .......................................................................................................... 38 2.2.5 Conformation population analysis ................................................................................ 39 2.2.6 Antibody labeling of SAGA ......................................................................................... 39 2.2.7 Chemical cross-linking and mass spectrometry analysis ............................................. 39 2.2.8 Expression and purification of recombinant transcriptional activators ........................ 40 2.2.9 Activator pulldown experiments and Western blotting ................................................ 41 2.2.10 Activator binding localization experiments ................................................................ 42 viii  2.2.11 Modeling ..................................................................................................................... 42 2.2.12 SAGA module localization ......................................................................................... 42 2.3 Results ................................................................................................................................. 43 2.3.1 Improved procedure for isolating native S. cerevisiae SAGA ..................................... 43 2.3.2 SAGA adopts three distinct conformations .................................................................. 43 2.3.3 Removal of key subunits affects SAGA’s conformational flexibility .......................... 47 2.3.4 3D reconstructions of SAGA in three conformations .................................................. 50 2.3.5 Tra1 occupies a substantial portion of SAGA’s head region ....................................... 52 2.3.6 Comprehensive EM-based mapping of subunit localization ........................................ 53 2.3.7 Cross-linking mass spectrometry analysis of apo-SAGA ............................................ 56 2.3.8 Interaction interface with transcriptional activators ..................................................... 59 2.3.9 Model of SAGA subunit arrangement in the context of the EM map .......................... 61 2.3.10 SAGA modules are transported to the nucleus individually ...................................... 65 2.4 Discussion ........................................................................................................................... 65 2.4.1 Conformational flexibility of SAGA ............................................................................ 67 2.4.2 Spatial arrangement of SAGA chromatin-binding domains......................................... 69 2.4.3 Site of SAGA assembly ................................................................................................ 71 Chapter 3: Modular organization of the essential yeast lysine acetyltransferase NuA4 ............... 73 3.1 Introduction ......................................................................................................................... 73 3.2 Experimental methods ......................................................................................................... 74 ix  3.2.1 S. cerevisiae strain construction ................................................................................... 74 3.2.2 Purification of NuA4 .................................................................................................... 74 3.2.3 NuA4 buffer optimization ............................................................................................ 76 3.2.4 Electron microscopy ..................................................................................................... 76 3.2.5 Image processing .......................................................................................................... 77 3.2.6 Chemical crosslinking coupled to mass spectrometry .................................................. 77 3.2.7 Modeling NuA4 architecture ........................................................................................ 77 3.2.8 Analysis of human TIP60 complex .............................................................................. 78 3.3 Results and discussion ......................................................................................................... 78 3.3.1 Purification of intact NuA4 from S. cerevisiae ............................................................ 78 3.3.2 Three dimensional reconstruction of NuA4 and subunit localization .......................... 79 3.3.3 Crosslinking coupled to mass spectrometry analysis ................................................... 84 3.3.4 Model of NuA4 subunit organization ........................................................................... 86 Chapter 4: Molecular architecture of the yeast Elongator complex ............................................. 91 4.1 Introduction ......................................................................................................................... 91 4.2 Experimental methods ......................................................................................................... 92 4.2.1 Yeast methods and strain construction ......................................................................... 92 4.2.2 Purification of yeast Elongator ..................................................................................... 92 4.2.3 Antibody labeling analysis ........................................................................................... 94 4.2.4 Electron microscopy ..................................................................................................... 95 x  4.2.5 Image processing .......................................................................................................... 95 4.2.6 Crosslinking coupled to mass spectrometry ................................................................. 97 4.2.7 Identification of cross-linking peptides and interacting proteins ................................. 98 4.2.8 Identification of co-purified proteins from Elongator subunit deletion strains ............ 98 4.2.9 Yeast phenotypic assays ............................................................................................... 99 4.2.10 Elp2 loop mutant copurification ................................................................................. 99 4.4.11 Construction of the multi-scale model of Elongator ................................................ 100 4.2.12 Overexpression and purification of recombinant Elp456 subcomplexes ................. 100 4.2.13 Malachite green ATPase assay ................................................................................. 101 4.2.14 Elongator and recombinant Elp456 mixing experiment ........................................... 101 4.2.15 Accession codes ........................................................................................................ 101 4.3 Results ............................................................................................................................... 101 4.3.1 Yeast Elongator adopts an asymmetric overall architecture ...................................... 101 4.3.2 Subunit connectivity of holo-Elongator ..................................................................... 108 4.3.3 Conserved loop regions of Elp2 are crucial to Elongator function ............................ 111 4.3.4 Model of yeast Elongator architecture ........................................................................ 113 4.3.5 Elp123 can accommodate two Elp456 subcomplexes ................................................ 115 4.4 Discussion ......................................................................................................................... 117 Chapter 5: Conclusions and future directions ............................................................................. 123 5.1 Module sharing between KATs and other chromatin-modifying complexes ................... 126 xi  5.2 The role of Elongator in the cell........................................................................................ 128 5.3 High resolution structural studies of KAT complexes ...................................................... 130 References ................................................................................................................................... 134 Appendices .................................................................................................................................. 163 Appendix A: Supplementary tables......................................................................................... 163 Appendix B: Publications arising from graduate work ........................................................... 208                    xii  List of tables Table 1.1. Chromatin reader domains found in lysine acetyltransferase complexes .................... 18 Table 1.2. Human homologues of the SAGA, NuA4, and Elongator complexes......................... 27 Table 2.1. Subunits of yeast SAGA .............................................................................................. 33 Table 2.2. S. cerevisiae strains used in this study of SAGA ......................................................... 35 Table 2.3. Mass spectrometry analysis of purified SAGA unit composition ............................... 45 Table 2.4. Validation of crosslinks detected in SAGA CXMS experiments ................................ 59 Table 3.1. Subunits of yeast NuA4 ............................................................................................... 74 Table 3.2. Strains used in this study of NuA4 .............................................................................. 75 Table 3.3. Validation of crosslinks detected in NuA4 CXMS experiments ................................. 86 Table 4.1. List of strains and plasmids used in this study of Elongator ....................................... 93 Table 4.2. Band quantification of Coomassie blue stained SDS-PAGE gel of full Elongator ... 103 Table 4.3. Validation of crosslinks detected in Elongator CXMS experiments ......................... 110 Table 4.4. Band quantification of Coomassie blue stained SDS-PAGE gel of full Elongator incubated with an excess of recombinant Elp456 ....................................................................... 119 Table A.1. Results from crosslinking mass-spectrometry of SAGA .......................................... 163 Table A.2. Results from crosslinking mass-spectrometry of NuA4 ........................................... 178 Table A.3. Results from crosslinking mass-spectrometry of Elongator ..................................... 192  xiii  List of figures Figure 1.1. Catalytic mechanism of GNAT enzymes ................................................................... 11 Figure 1.2. Catalytic mechanism of MYST enzymes ................................................................... 15 Figure 1.3. Saccharomyces cerevisiae lysine acetyltransferases (KATs) and their subunits. ...... 19 Figure 2.1. Purification of S. cerevisiae SAGA and negative stain EM analysis. ......................... 44 Figure 2.2. Effects of mutations on SAGA flexibility. ................................................................. 48 Figure 2.3. Three-dimensional reconstructions of the three SAGA conformations. .................... 51 Figure 2.4. Localization of SAGA domains and subunits. ........................................................... 54 Figure 2.5. Network of SAGA subunit interconnectivity determined using cross-linking mass spectrometry. ................................................................................................................................. 58 Figure 2.6. Localization of transcriptional activator binding sites. .............................................. 60 Figure 2.7. Spatial arrangement of SAGA subunits. .................................................................... 62 Figure 2.8. Initial investigation of SAGA module localization .................................................... 66 Figure 2.9. Chromatin-binding domains of SAGA cluster around one edge of the complex. ...... 70 Figure 3.1. Purification and visualization of yeast NuA4. ............................................................ 80 Figure 3.2. Optimizing NuA4 purification buffers ....................................................................... 81 Figure 3.3. Mapping of NuA4 lobes and subunits. ....................................................................... 82 Figure 3.4. 3D reconstruction of NuA4. ....................................................................................... 83 Figure 3.5. Crosslinking coupled to mass spectrometry of NuA4. ............................................... 85 Figure 3.6. Model of NuA4 subunit organization. ........................................................................ 87 xiv  Figure 3.7 Initial EM analysis of the human Tip60 complex ....................................................... 89 Figure 3.8. 3D reconstruction of TIP60. ....................................................................................... 90 Figure 4.1. Elongator is a two-lobed asymmetric complex ........................................................ 102 Figure 4.2. Negative stain class averages of intact Elongator and the Elp123 subcomplex ....... 104 Figure 4.3. 3D reconstruction of the Elongator complex ............................................................ 107 Figure 4.4. Mass spectrometry analysis of Elongator ................................................................. 109 Figure 4.5. Conserved Elp2 loops are important for Elongator function.................................... 112 Figure 4.6. Model of Elongator subunit organization and Elp456 association ........................... 112 Figure 4.7. 3D reconstruction of the Elp123 subcomplex .......................................................... 116 Figure 4.8: Elongator can accommodate two copies of the Elp456 subcomplex ....................... 118 Figure 4.9. Investigating loading two copies of Elp456 on the Elongator complex ................... 120           xv  List of abbreviations Acetyl-CoA Acetyl coenzyme A AD  Activation domain ADA  Transcriptional adaptor ATAC  Ada2A-containing ATP  Adenosine triphosphate CBP/p300 Creb-binding protein/p300 CCD  Charge-coupled device COMPASS Complex of proteins associated with Set1 CTD  C-terminal domain CTF  Contrast transfer function CXMS  Crosslinking coupled to mass spectrometry DNA  Deoxyribonucleic acid DSS  Disuccinimidyl suberate DUB  Deubiquitination EM  Electron microscopy FACT  Facilitates chromatin transcription FAT  FRAP-ATM-TRRAP FATC  C-terminal FRAP-ATM-TRRAP FRB  FKBP12-rapamycin binding FSC  Fourier shell correlation GFP  Green fluorescent protein GNAT  Gcn5-related N-acetyltransferase GraFix  Gradient fixation H_K_  Histone _ lysine _ HBO1  Histone acetyltransferase binding to Orc1 HEAT  Huntingtin, EF3, PP2A, Tor1 HMP  His-maltose binding protein xvi  HSA  Helicase SANT-associated IKBKAP Inhibitor of Kappa light polypeptide gene enhancer in B-cells Kinase complex Associated Protein KAT  Lysine (K) acetyltransferase KDAC  Lysine deacetylase LC-MS/MS Liquid chromatography coupled with tandem mass spectrometry MBP  Maltose binding protein mcm5  5-methoxycarbonylmethyl Me  Methylated MgOAc Magnesium acetate MOZ/MORF Monocytic leukemia zinc finger protein/MOZ-related factor) mRNA  Messenger RNA MSL  Male-specific lethal mTOR  Mammalian target of rapamycin MYST  MOZ, Ybf2, Sas2, Tip60 ncm5  5-carbamoylmethyl NFR  Nucleosome-free region NSL  Non-specific lethal NuA4  Nucleosomal acetyltransferase of histone H4 PCAF  p300/CBP-associated factor PIKK  Phosphatidylinositol 3-kinase-related kinase Pol II  RNA polymerase II PTM  Post-translational modification RNA  Ribonucleic acid RPD3S Reduced Potassium Dependency 3 complex-Small RSC  Remodel the structure of chromatin SAGA  Spt-Ada-Gcn5 acetyltransferase SAM  S-adenosylmethionine SANT  Swi3, Ada2, N-Cor, and TFIIIB xvii  STAGA  Spt-Taf9-Ada-Gcn5 acetyltransferase SWI/SNF Homothallic switching deficient/sucrose non-fermenting SWIRM Swi3, Rsc8, and MOIRA SWR1C Swi2/Snf2-related 1 complex TAP  Tandem affinity purification TBP  TATA binding protein TCA  Trichloroacetic acid TEV  Tobacco Etch Virus TFIID  Transcription factor IID TIP60  HIV-1 Tat-interactive protein, 60 kDa TPR  Tetratricopeptide repeat TREX-2 Transcription export 2 tRNA  Transfer RNA TSS  Transcription start site Ub  Ubiquitinated USP  Ubiquitin-specific protease YEATS Yaf9, ENL, AF9, Taf14, and Sas5        xviii  Acknowledgments  First and foremost I would like to thank my family for their unwavering support of my education and career path, and my future wife Perdita Wito for her unending patience and understanding.  I am also extremely grateful to my supervisor Dr. Calvin K. Yip for providing me the opportunity to embark upon this wonderful endeavour of academic research. He supported my development as a scientist, granting me the independence to pursue research paths that range from promising to mildly ludicrous. Dr. Yip has, without hesitation, sent me to conferences around the world to learn new techniques and enrich my professional development. He is my first and foremost mentor in science, and for that I will forever be in his debt. I would also like to thank my committee members Dr. Natalie Strynadka and Dr. LeAnn Howe for their guidance throughout my PhD studies.  Much of my research would not have been possible without valuable assistance and communication with my colleagues. Every member of the Yip lab past and present have been instrumental in my studies through productive discussions and technical help. I’ve had the distinct pleasure to be assisted by many undergraduates: J.R., H.R., D.C., C.L., and J.T., and hopefully their experiences have helped them in their careers. I would also like to thank Dr. Shan Lu and Dr. Meng-Qiu Dong for being valued collaborators in all of my research endeavours.  I am also very grateful to the Natural Sciences and Engineering Research Council of Canada for scholarship support.  1  Chapter 1: Introduction  Humanity has recognized that traits are passed down from parent to child for thousands of years. We have exploited heredity almost instinctively, using selective breeding to craft a staggering variety of livestock and crops nearly unrecognizable from their wild progenitors. In the middle of the 19th century, an Augustinian friar named Gregor Mendel identified that hereditary traits occur in discrete units: genes. Genes from parents are transferred to their children, and their combinations determine the traits that the children display. It took another century, however, until the molecular nature of these genes were characterized.  Although the deoxyribonucleic acid (DNA) polymer was discovered in the 19th century, its role in genetic inheritance was not confirmed until 1943 through a series of experiments conducted by Avery, MacLeod, and McCarty1. The Avery-MacLeod-McCarty experiment showed that DNA, not ribonucleic acid (RNA) or proteins, can confer traits from one strain of bacteria to another. Soon after, work by Watson, Crick, and Franklin revealed that DNA is a giant ladder-like polymer consisting of two polynucleotide strands, whose ‘rungs’ were formed by pairs of complementary bases2. Critically, these two strands can be separated to expose the bases, therefore providing a template for the original sequence of bases to be faithfully recapitulated. In 1958, Crick correctly postulated the ‘central dogma of biology’, where DNA sequence determines RNA (a similar but less stable molecule to DNA) sequence, which in turn determines the sequence of amino acids that constructs the primary machinery of the cell: proteins.  The discovery of DNA and its roles in genetic inheritance and expression of certain traits (phenotype) raised an important question: if every cell in a multicellular organism contains the same DNA sequence, what differentiates skin cells from brain cells and other types of cells, 2  whose phenotypes are wildly different from their progenitor embryonic cells? Organisms express different genes based on its environmental and temporal contexts through a variety of mechanisms, such as regulating its gene transcription regime.  One such mechanism of transcription regulation is chromatin, an exquisitely regulated structure consisting of proteins and DNA that affects when, where, and how much a certain gene is expressed.  1.1 Organization of eukaryotic chromatin  Eukaryotic chromatin consists of DNA wrapped around protein complexes called nucleosomes, which is an octamer of histone proteins. Early studies found that nucleosomes stud the entire DNA strand at regular intervals to form a structure resembling beads on a string3–5. These beads associate with each other to form even higher orders of organization and packing DNA to progressively more compact structures6. As intuition would suggest, wrapping DNA into tightly packed nucleoprotein structures restricts its accessibility to sequence-reading cellular machinery. Critically, RNA Polymerase II (Pol II), the enzyme responsible for transcribing messenger RNA from DNA, is generally inhibited by the presence of nucleosomes in the vicinity of its target genes7, while depleting nucleosomes stimulates transcription8,9. Regions of the genome that are transcriptionally silent form a highly packaged form of chromatin called heterochromatin. Conversely, transcriptionally active chromatin, euchromatin, is more loosely arranged. Chromatin structure therefore serves as a major determinant for transcription.  Individual nucleosomes consist of two copies each of four core histone proteins: H3, H4, H2A, and H2B10–13, which is wrapped by approximately 146 base pairs of DNA. Histones are positively-charged proteins, a feature that facilitates its strong interactions with the negatively charged DNA phosphate backbone. Histones H3 and H4, as well as H2A and H2B form dimers. Two H3-H4 dimers then form a tetramer, and two H2A-H2B dimers associate with this tetramer 3  to form the nucleosomal octamer. DNA is wound approximately 1.65 times around this octamer, forming numerous interactions with its constituent histones The histones’ N-termini form largely unstructured tails that contact the nucleosomal DNA while remaining accessible to the solvent. Nucleosomes can act as steric barriers against transcriptional machinery, necessitating their depletion at the site of transcription.  Genome-wide studies of nucleosome positioning found an irregular pattern of nucleosome occupancy in the vicinity of genes within euchromatin14,15. The DNA sequence directly upstream of gene transcription start sites (TSS) are described as promoters and act as an assembly site for transcription machinery. Promoters contain a region that is often depleted of nucleosomes15. These regions, named nucleosome free regions (NFR), serve as the major binding sites for transcription factors, which are sequence-specific DNA-binding proteins that facilitate initiation of transcription16. Nucleosomes that flank the NFR are the most consistently positioned within a population, and nucleosomes further from the NFR get progressively ‘fuzzier’14. Despite the imprecise positioning of nucleosomes found in the gene coding region 3’ of the NFR and TSS, they are still present and must be addressed for RNA Pol II to traverse the entire length of the gene.  Many classes of proteins regulate nucleosome positioning and occupancy. These chromatin regulators respond to specific stimuli and work together to formulate the appropriate response. Since any cellular process that requires access to DNA necessarily interfaces with chromatin, these regulators are central to normal cellular function. This introduction aims to provide a brief overview of several topics intimately related to these regulators and the cellular mechanisms that they facilitate. Section 1.2 explores how transcription is influenced by chromatin structure and post-translational modification of histones. We delve further into the 4  mechanisms behind chromatin regulation in section 1.3 by discussing several protein domains and motifs that interact with and modify nucleosomes. Finally, section 1.4 assembles these domains into their final forms in vivo: huge, multiprotein, chromatin-modifying complexes. 1.2 Transcription and chromatin in yeast  The discovery of nucleosomes and their role as gatekeepers to DNA ignited a massive drive towards characterizing the interface between DNA-related processes and chromatin. Fundamental cellular processes such as transcription involves constructing a huge protein complex at the transcription start site that bulls its way through the gene, prying the two DNA strands apart that only moments ago were tightly wound around nucleosomes17. Not only do nucleosomes in the path of transcriptional machinery yield to it and re-establish in its wake18, changes in chromatin also help to incite transcription in response to environmental stimuli16. How transcription interact with and alter the chromatin landscape will be further discussed in this section. The budding yeast Saccharomyces cerevisiae emerged as an important model organism for analyzing these chromatin-associated processes. Nucleosomes and many vital chromatin-modifying factors are strongly conserved from yeast to humans19. These similarities, combined with the ease of genetic manipulation of its genome, made S. cerevisiae a powerful platform for chromatin experimentation. From biochemical isolation of the first chromatin-modifying protein complexes to clever genetic screens, work done in yeast has spearheaded multiple avenues of research in the field16. In fact, several groundbreaking genetic screens conducted in yeast were the first experiments that identified chromatin as a key player in transcription regulation20–22. The advantages of working in this model organism remain to this day, and this introduction will focus on both historical and contemporary research conducted in yeast. 5   Transcription serves as the first step of regulating expression of protein-coding genes: DNA template is read by transcriptional machinery—primarily the RNA polymerase II complex—and transcribed into mRNA, which is then exported from the nucleus and translated into proteins. At any given moment in a cell’s life, thousands of genes are potentially being transcribed. These different transcriptional regimes are often accompanied and facilitated by alterations in chromatin structure. One important axis of regulation for chromatin structure is the post-translational modification of histones. 1.3 Histone post-translational modifications  Post-translational modification of histones play critical roles in transcription regulation. Histone post-translational modifications (PTMs) are various functional molecules that are covalently attached to histones, and can range from small chemical groups to entire proteins16. These modifications have profound impacts on chromatin arrangement and serve as docking sites for many chromatin-interacting proteins16. This dissertation will focus on histone acetylation and the enzymes that catalyze its deposition, lysine acetyltransferases (KATs). Intriguingly, KATs often occur in the context of large multisubunit complexes that interact with other histone modifications23. This section discusses three different histone PTMs that intersect with the activity of KAT complexes: acetylation, methylation, and ubiquitination. 1.3.1 Histone acetylation The major PTM associated with transcription activation is histone acetylation, where an acetyl group is added to the ε-amine of lysine residues. These modifications are deposited by lysine acetyltransferase enzymes (KATs), and removed by lysine deacetylase enzymes (KDACs). Histone acetylation is highly enriched in active gene promoters and correlates strongly with increased rates of transcription24,25. Although the amount of histone acetylation 6  observed decreases in the coding region of genes, KATs still play an active role in these sites26. KATs are present in the coding region of genes, and their acetyltransferase activities stimulate the elongation of RNA and nucleosome eviction in gene bodies26,27.  Histone acetylation activates transcription through two proposed mechanisms: neutralization of the positive charges in histone tails and recruitment of other effectors such as chromatin remodelers. Acetylation of the lysine side chain neutralizes its positive charge, which directly weakens the nucleosome’s interaction with the negative DNA phosphodiester backbone28. Acetylated histones also serve as binding sites for proteins that possess bromodomains—an approximately 110 amino acid module that specifically binds acetylated lysines29—that can further modify the chromatin landscape. For example, the bromodomain-containing chromatin remodeling complexes RSC (Remodel the Structure of Chromatin) and SWI/SNF (homothallic SWItching deficient/Sucrose NonFermenting) are both recruited by acetyl marks on histones30–32. Chromatin remodelers can alter the nucleosomal architecture at promoters to facilitate transcription factor binding and subsequent transcription initiation33–35. Through both its physical effects on individual nucleosomes to its role in recruiting chromatin remodelers, histone acetylation is an important activator of transcription. 1.3.2 Histone methylation Lysines in histone tails are also subject to methylation by histone methyltransferase enzymes. In S. cerevisiae, lysines 4, 36, and 79 of histone H3 can be mono-, di-, or trimethylated36. Unlike histone acetylation, histone methylation does not affect the lysine positive charge. Instead, its effects are mediated through proteins containing one of the many different types of domains that specifically recognize methylated histones37. One notable aspect of yeast histone methylation is the lack of association with heterochromatin. In higher 7  eukaryotes, H3K9 methylation is a well conserved mark that silences transcription and forms heterochromatin through association with the HP1 protein38,39. However, all three yeast methylation marks are associated with actively transcribed genes.  Methylation of histone H3 at lysine 4 (H3K4) is catalyzed by Set1 and is found throughout the length of a gene and its promoter25. The type of H3K4 methylation follows a gradient based on its location in the gene: trimethylation dominates the 5’ end, monomethylation the 3’ end, and dimethylation between them25. Set1, found within the COMPASS (COMplex of Proteins ASsociated with Set1) complex, associates with Pol II in the process of elongating through the gene to deposit methyl marks to its target histones40. H3K4 methylation recruits a number of other histone-binding proteins with various effects on transcription41,42 Methylation of H3K36 is catalyzed by Set2 and is enriched throughout the gene body, especially towards the 3’ end25,43. Like Set1, Set2 methylation is guided by the elongating RNA Pol II complex as it transcribes through a gene44. This mark is specifically recognized by Eaf3, a methyllysine-binding protein found in both the NuA4 (Nucleosomal Acetyltransferase of histone H4) KAT and RPD3S (Reduced Potassium Dependency 3 complex-Small) KDAC complexes45. NuA4 recruitment through H3K36 methylation results in acetylation of histone H4, a mark associated with transcription activation45. Conversely, RPD3S deacetylation suppresses spurious intragenic transcription that often occurs as a byproduct of transcription elongation46.  Finally, H3K79 is monomethylated by Dot1 within gene bodies25,47. Studies in mammalian cells found Dot1 in complex with proteins involved in stimulating transcription elongation, suggesting a link between H3K79 methylation and elongation48. More recently, H3K79 methylation has been shown to interact with H2BK123 ubiquitination (H2BK123Ub). Histone ubiquitination will be further discussed below, but its presence on H2BK123 stimulates 8  the deposition of both H3K4 and H3K79 trimethylation marks49. The removal of H2BK123Ub depends on the methylation state of its host nucleosome, with deubiquitinases Ubp8 and Ubp10 targeting regions enriched in H3K4 and H3K79 trimethylation, respectively49. Histone methylation’s effects on transcription and their crosstalk with other histone modifications remains an intriguing topic of study. 1.3.3 Histone ubiquitination  In addition to small chemical modifications, histones can also be conjugated to a single ubiquitin protein50. Although ubiquitination is usually associated with polyubiquitination of proteins destined for degradation by the proteasome51, non-degradative monoubiquitination of histones plays a variety of roles in transcription50. Monoubiquitination of histone H2B lysine 123 is catalyzed by Rad6-Bre1, a complex of an E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase, and is enriched in gene bodies52,53. Although the H2BK123Ub mark increases the stability of nucleosomes, it can both activate and suppress transcription54–56. In particular, H2BK123Ub stimulates transcription in highly expressed genes54,55. In this context, nucleosome stabilization may facilitate transcription by reassembling the chromatin template in the wake of Pol II passage through interactions with the histone chaperone FACT (FAcilitates Chromatin Transcription)57. Conversely, H2BK123Ub represses transcription in poorly transcribed genes54. RNA Polymerase II does not constitutively associate with these genes, and the stabilized nucleosomes may prevent its recruitment to these loci50,54.  Independently of nucleosome stabilization, H2BK123Ub also participates in transcription elongation. Different phases of transcription elongation is defined by the phosphorylation state of the C-terminal domain (CTD) of the largest subunit of Pol II, which contains many repeats of the amino acid sequence YSPTSPS58. Phosphorylation of the fifth serine of the CTD repeat by 9  Kin28 triggers transcription elongation and is required for H2BK123 ubiquitination58,59. In the middle of the gene, Ctk1 phosphorylates the second serine of the CTD repeat to mark the next phase of transcription elongation58. The presence of H2BK123Ub prevents Ctk1 recruitment, and its removal by the SAGA subunit Ubp8 permits the transition towards the next phase of elongation60.   Finally, H2BK123Ub is intimately linked to histone methylation, being required for both H3K4 and H3K79 methylation61. This ubiquitin mark stimulates the ubiquitination of the Swd2 subunit of COMPASS, which in turn activates the complex’s H3K4 methyltransferase activity62. Swd2 also physically interacts with Dot1, and may regulate H3K79 methylation through the same mechanism50,61. 1.4 Protein domains found in lysine acetyltransferases  KAT complexes, especially larger ones, are highly complicated molecular machines involved in many different pathways regulating transcription23. For example, the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the primary histone H3 acetyltransferase in yeast, also recruits the general transcription factor TBP (TATA-Binding Protein) to promoters, deubiquitinates histone H2B, and shares subunits with mRNA export machinery63. However, even the most complex machines are constructed from simple parts and modules, each with a specific function, that cooperate to accomplish seemingly insurmountable feats. This section focuses on the individual parts found in KAT complexes, and how their structures facilitate their functions.  1.4.1 Histone acetyltransferase domains  Histone acetyltransferases use acetyl-CoA as a source of acetyl groups to transfer to the acceptor histones. These enzymes are conserved from yeast to humans and often form large 10  complexes with other proteins23. These enzymes can be categorized into two main families: the GNAT (Gcn5-related N-AcetylTransferase) and the MYST (MOZ, Ybf2, Sas2, Tip60) families23. Recent research have shown multiple examples of histone acetyltransferases specifically acetylating non-histone substrates to modulate their functions64,65, and histone acetyltransferases will be referred to in this dissertation as lysine acetyltransferases (KATs) accordingly. This section will discuss the structure and function of the two largest KAT families: GNAT and MYST. GNAT family  The Gcn5-related N-acetyltransferase (GNAT) is a large and highly conserved family of KATs66. The catalytic domain of GNATs is characterized by either six or seven beta strands and four alpha helices66. The secondary structure arrangement is: β0-β1-α1-α2-β2-β3-β4-α3-β5-α4-β6 (Fig 1.1A-B)66. The N-terminal motifs (β0-β1-α1-α2-β2-β3) are important for the stability of the fold. Strands β4 and β5 form the acetyl-CoA binding site, while β1, β2, α4, and β6 bind the acceptor substrate. The loop between strand β4 and helix α3 is termed the P-loop and binds the pyrophosphate of acetyl-CoA. A beta bulge formed by β4 and β5 forms an oxyanion hole that stabilizes the tetrahedral intermediate of the acetylation reaction. Despite low sequence conservation, this structural organization is largely conserved between members of the family66. GNAT enzymes have a conserved ordered bi-bi catalytic mechanism (Fig 1.1C)67,68. The acetylation target, a primary amine, is deprotonated by a general base near the active site. The deprotonated amine then conducts a nucleophilic attack on acetyl-CoA to form a tetrahedral intermediate. The ternary complex is then broken down when a general acid protonates the CoA group while a general base deprotonates the acetyl-amine group. Despite the structural and catalytic conservation between GNAT family enzymes, their specific cellular roles and binding 11  partners introduce an element of variability to their functions. Here we discuss the GNAT family members Gcn5 and Elp3.  Figure 1.1. Catalytic mechanism of GNAT enzymes. (A) X-ray crystallographic structure of Tetrahymena thermophila Gcn5 in complex with coenzyme A (CoA) and a histone H3 peptide (PDB ID: 1QSN69). (B) Arrangement of catalytic residues in the active site. (C) Proposed catalytic mechanism of Gcn5: An active site glutamate acts as a general base and deprotonates the target lysine ε-N through a water molecule. The deprotonated amine nucleophilically attacks the carbonyl carbon of acetyl-CoA, forming a tetrahedral oxyanion intermediate. The intermediate collapses to release CoASH and the acetylated lysine.  12 Gcn5 and the histone acetyltransferase module  Gcn5 is one of the most well characterized KAT, with available X-ray structures of the catalytic site from Tetrahymena thermophila and Saccharomyces cerevisiae enzymes, and the human homologue PCAF69–71.  Yeast Gcn5 contains five alpha helices and six beta strands arranged overall in the canonical GNAT fold70. The core region of its catalytic site is highly conserved with other GNAT enzymes, but its N and C termini are divergent. In particular, these divergent regions—α1-α2, β5-α4, α5-β6 pairs—are responsible for H3 peptide binding69. The P-loop of Gcn5 is formed by the β4-α3 pair, and is likely stabilized by CoA binding70. An active site glutamate acts as the general base that deprotonates its primary amine target; in this case the H3 lysine68. Detailed kinetic analysis of PCAF confirmed that the enzyme utilizes an ordered bi-bi mechanism with acetyl-CoA binding preceding H3 loading, with a transient enzyme-acetyl-CoA-H3 ternary complex being formed72. These results reaffirm Gcn5’s identity as a bona fide GNAT family acetyltransferase.  Intriguingly, Gcn5 by itself cannot acetylate nucleosome substrates73. Recombinant Gcn5 has modest acetylation activity for H3K14 and H4K8 lysines, which is distinct from its in vivo acetylation of nucleosomal histones H3 and H2B74,75. Later research discovered that native Gcn5 occurs in complex with multiple other subunits in either the SAGA or ADA (transcriptional ADAptor) complexes75. In particular, SAGA Gcn5 belongs to a 4-subunit histone acetyltransferase module (Gcn5, Ada2, Ada3, and Sgf29) within the 19 subunit complex, while the entire ADA complex consists of Gcn5, Ada2, Ada3, Sgf29, and two unique subunits: Ahc1 and Ahc263,75. Purified SAGA acetylates nucleosomal H3K9, H3K14, H3K18, H3K23, and histone H2B, while ADA acetylates nucleosomal H3K14 and H3K1874. These findings highlight the importance of the molecular context of Gcn5 in determining its acetylation targets. 13 Elp3, a modified GNAT  The Elp3 subunit of Elongator has been shown to contain a KAT domain of the GNAT family76. Intriguingly, it also contains a radical S-adenosyl methionine (SAM) domain which uses an iron sulfur cluster to generate radical intermediates in the catalysis of reactions such as methylation77. Although Elp3’s role as a bona fide histone acetyltransferase has not been unequivocally established, multiple experiments show that it is involved in the 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) modifications of transfer RNA (tRNA) wobble base pair78–80.  A recent crystal structure of archaeal Elp3 showed that the KAT domain folded similarly to other GNAT members, with the addition of a seventh beta strand and the absence of helices α1 and α281. Strangely, the N and C termini of the domain, where histone peptides would bind in the homologous Gcn5 structure, is fully occluded by the radical SAM domain. Elp3 still binds acetyl-CoA in a tRNA-stimulated manner82. Based on the crystal structure of Elp3 and a mass spectrometric analysis of its catalytic products, the following catalytic mechanism was proposed81,82: S-adenosyl methionine binds the radical SAM domain to generate the 5’-deoxyadenosyl radical, followed by the binding of tRNA and acetyl-CoA. The radical facilitates the conjugation of the acetyl-CoA with the tRNA through a C-C bond. Water then acts as the nucleophile to hydrolyze and release CoA and the modified tRNA. This mechanism ostensibly retains the ordered bi-bi ‘acetylation’ of other GNAT enzymes (except the ‘acetyl group’ is attached to the tRNA and the nucleophile is water instead of a primary amine), and may be the first example of the adaptation of the GNAT catalytic mechanism as part of a more complex pathway.  14 MYST family and Esa1 The MYST family acetyltransferases are named after its founding members: MOZ, Ybf2 (Sas3), Sas2, and Tip6083. The overall secondary structure of MYST enzymes is as follows: β1-β2-β3-β4-α1-β5-β6-α2-β7-β8-β9-α3- β10 (Fig 1.2A)84,85. Despite the lack of sequence conservation with GNAT enzymes, the core CoA-binding region and the P-loop (β7-β8-β9-α3) adopt a similar structure (Fig 1.2B)83. The β3-turn-β4-α1 region forms the zinc finger commonly found in MYST type KATs84,85. However, only three MYST enzymes have been crystallized (PDB IDs: 1MJA84, 2RC485, and 4GIV), likely due to their large size. More structural analysis to this group of enzymes will help elucidate the conserved structure of the catalytic site. Despite the similarities of GNAT and MYST core structures, their catalytic mechanisms are distinct. The initial deprotonation of the primary amine target likely occurs analogously, as Esa1 contains an essential glutamate residue oriented towards the active site84. However, Esa1 can carry a transferable acetyl group through a conserved cysteine near its acetyl-CoA binding site not found in GNAT enzymes84,86. These observations suggest that Esa1 (and other MYST enzymes) utilizes a ping-pong reaction mechanism, where acetyl-CoA first binds the enzyme and transfers the acetyl group to the conserved cysteine (Fig 1.2C). Then the acceptor substrate is bound and accepts the acetyl group, and subsequently released. Interestingly, recent research found that MYST enzymes require autoacetylation at a conserved active site lysine for function in vitro and in vivo87. Similar to Gcn5, Esa1 cannot acetylate nucleosomal templates without being either part of a KAT module in NuA4, or as the catalytic subunit of the four subunit Piccolo complex88,89. The overall characteristics of MYST structure and function gives the impression of a more specialized KAT compared to GNAT enzymes, and the divergence  15  Figure 1.2. Catalytic mechanism of MYST enzymes. (A) Structure of Saccharomyces cerevisiae Esa1 (PDB ID:3TO784). Conserved secondary structure elements are shown. (B) Comparison of the coenzyme A (CoA) binding region of tGcn5 and yEsa1 (β7-β8-β9-α3). (C) Proposed catalytic mechanism of MYST enzymes: An active site cysteine is deprotonated by a glutamate residue acting as a general base and the γ-thiol conducts a nucleophilic attack on the carbonyl carbon of acetyl CoA, forming a tetrahedral intermediate. The intermediate collapses, transferring the acetyl group to the active site cysteine, and CoASH is released. The acceptor lysine is then deprotonated by the active site glutamate, and nucleophilically attacks the acetylated cysteine. The intermediate collapses to release an acetylated lysine residue. 16  between these two groups in both structure and chromatin regulation is an intriguing evolutionary event. 1.4.2 Ubp8 histone deubiquitinase domain  Non-degradative monoubiquitination of histones has increasingly been shown to participate in regulating chromatin90. As previously discussed, ubiquitin removal is an important aspect of progress through the transcriptional elongation phase. Deposited ubiquitin groups are removed through the action of deubiquitinating enzymes (DUBs), such as the SAGA subunit Ubp856. Ubp8 is a DUB from the USP (Ubiquitin Specific Protease) family, whose members utilize a catalytic Cys-His-Asp/Asn triad to hydrolyze the peptide bond between proteins and ubiquitin91,92. The active site cysteine is deprotonated by an adjacent histidine residue and polarized by an aspartate or asparagine. The thiol group then conducts a nucleophilic attack on the carbonyl carbon of the target peptide bond to form an oxyanion intermediate. The collapse of the oxyanion intermediate releases the protein, leaving ubiquitin bound to the enzyme. A water molecule then attacks the cysteine-ubiquitin bond and results in the release of the enzyme and free ubiquitin. Despite a relatively simple catalytic mechanism, USP catalytic sites have a large and characteristic structure.  USP domains fold into a thumb-palm-fingers structure92. Aside from insertions at several loop regions, the core USP domain consists of sixteen beta strands and eight alpha helices92. The thumb and the palm are stacked on each other, while the fingers project outwards away from the centre of the domain. The active site is formed between the thumb and palm, and the ‘fingertips’ form a zinc binding site. Specificity to the globular ubiquitin substrate is mediated by the fingers. Ubp8 adopts the same overall structure, except for two additional zinc binding motifs that 17  coordinate structural zinc ions93. However, similar to the Gcn5 and Esa1, Ubp8 occurs in the context of a catalytic module.  Ubp8 is found as part of the SAGA complex within a deubiquitination module consisting of Ubp8, Sgf73, Sgf11, and Sus193,94. The deubiquitinating activity of Ubp8 is greatly stimulated upon integration into the module93. Astounding crystal structures of the DUB module by itself and bound to an intact nucleosome octamer were recently published by the Wolberger group93,94. These structures showed that the other DUB module subunits serve to reinforce the shape of the catalytic USP domain of Ubp8. Furthermore, the Sgf11 subunit also contacts histone H2B, which may help mediate the specificity of the module94. The DUB module of SAGA further follows the trend of catalytic subunits within KAT complexes that are modulated by binding partners within a functional module. 1.4.3 Chromatin readers The previous section illustrated the role of modules in determining the specificity of KAT and DUB enzymes. However, Gcn5, Ubp8, and Esa1 modules are components of even larger complexes. Many of the subunits within these complexes contain domains that bind chromatin, and may serve to recruit the complex and their associated catalytic activities to specific nucleosomes23. Table 1.1 shows the chromatin reader domains found in these complexes, as well as their chromatin targets (PDB IDs: 2R0S, 2DY7, 2PNX, 3MP8, 3FK3, 4XFV)29,95–99,37,100–107,30,108–113. This section discussed multiple functional components found in lysine acetyltransferase complexes. Although KAT complexes are defined by their acetyltransferase activity, they also contain multiple other functional domains. Many of these domains specifically bind chromatin, with many of them recognizing a specific histone modification. As observed in Gcn5 and Esa1 18  acetyltransferases, these enzymes function in the context of a module with a handful of other subunits. Intriguingly, these modules also associate with multiple other modules to construct extremely large complexes such as the SAGA and NuA4 KATs. In contrast to Esa1 and Gcn5,        Table 1.1. Chromatin reader domains found in lysine acetyltransferase complexes 19  Elp3 contains an adapted GNAT domain that facilitates tRNA modification. Despite archaeal Elp3 being functional by itself, eukaryotic Elp3 requires five additional subunits for efficient activity114. How and why these proteins associate into sprawling complexes is an intriguing question that remains unanswered. 1.5 Lysine acetyltransferase complexes  The preceding section explored the functional modules found in lysine acetyltransferases. From a reductionist perspective, a transcriptional coactivator KAT complex should require only the catalytic module equipped with a chromatin reader domain, as well as some means to interact with transcriptional activators for recruitment to its target genes. Why then do they sometimes assemble into large multisubunit complexes (Fig 1.3)? This section will break down three lysine acetyltransferase complexes into their components and discuss their varied cellular roles.  Figure 1.3. Saccharomyces cerevisiae lysine acetyltransferases (KATs) and their subunits. (A) GNAT family KAT complexes. Spt7* is a C-terminally truncated form of Spt7. (B) MYST family KAT complexes. Catalytic subunits are in bold.  20  1.5.1 Spt-Ada-Gcn5 Acetyltransferase complex (SAGA)  The SAGA complex is the largest yeast KAT at 2 MDa and 19 subunits63. SAGA is a transcriptional coactivator—a complex that works with DNA-sequence specific transcription activator proteins32—responsible for activating transcription of stress-response genes115. In addition to being an acetyltransferase through its Gcn5 subunit, it is a deubiquitinase through its Ubp8 subunit. SAGA subunits can be split into four structural and functional modules: the lysine acetyltransferase (KAT), deubiquitinase (DUB), SPT (named after the majority of its subunits), and TAF (also named after its subunits) modules116. The KAT and DUB modules were previously discussed, and are responsible for SAGA’s catalytic activities. The SPT module subunits are responsible for maintaining SAGA structure, facilitating its recruitment to target genes, and interacting with TBP63,117–119. The TAF module contains subunits shared with the TFIID complex, and is thought to also serve an architectural role in SAGA63. Each of these modules contain characteristic building blocks that facilitate their functions.  The KAT module of SAGA consists of Gcn5, Ada2, Ada3, and Sgf29116. Gcn5 contains a bromodomain and a GNAT domain that facilitates the module’s acetyltransferase activity. Sgf29 contains a tandem Tudor domain that binds di- and trimethylated histone H3K4, serving as another means for SAGA recruitment in addition to transcriptional activator binding110. Ada2 houses an N-terminal ZZ-type zinc finger of unclear function, a SANT (Swi3, Ada2, N-Cor, and TFIIIB) domain which stimulates Gcn5 acetylation of nucleosomal templates, and a DNA-binding SWIRM (Swi3, Rsc8, and MOIRA) domain106,120. The module is anchored to the rest of SAGA through Ada2 and Ada3121. In addition to nucleosomes, Gcn5 also acetylates various nonhistone targets such as the ribosomal protein Ifh1 and the histone deacetylase complex subunit Ume6122,123.  21   The DUB module of SAGA is composed of Ubp8, Sus1, Sgf11, and Sgf7363. The USP domain of Ubp8 imparts the module’s deubiquitinase activity. Ubp8, Sgf11, and Sgf73 all contain zinc-binding domains that most likely maintain the structure of the module93. In addition to deubiquitinating histone H2B, Ubp8 also deubiquitinates the adenosine monophosphate (AMP)-activated kinase Snf1 to maintain its stability and activity124. Sgf73 anchors the entire module to SAGA. Recent work in human cells found that Sgf11 and Sus1 both activate other deubiquitinases independently of SAGA and Ubp8125. The Sus1 subunit is also an integral part of the nuclear pore-associated TREX-2 (TRanscription/EXport 2) complex that mediates efficient mRNA export from the nucleus126. Intriguingly, Sgf73 is required for integration of Sus1 to TREX-2 despite not being part of the complex127.   The SPT module of SAGA consists of Tra1, Ada1, Spt3, Spt8, Spt7, and Spt20116. Spt7 contains a bromodomain and a histone fold domain63. The histone fold domain, which Ada1 and Spt3 also possess, can associate with other histone folds akin to the nucleosome octamer128. Spt7, Spt20, and Ada1 are all important for the integrity of the SAGA complex, outlining the structural importance of the SPT module129. At approximately 400 kDa, Tra1 is SAGA’s largest subunit and is a catalytically inactive member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family130. The Tra1 subunit is responsible for association with transcriptional activators to recruit SAGA to its target promoters117. In addition to complex recruitment, the C-terminus of Tra1 is important for the structural integrity of SAGA130. Spt8 contains a WD40 domain and, along with Spt3, associates with TBP and facilitates its binding to SAGA-dependent promoters118,119. Interestingly, yeast possesses an alternate form of SAGA called SALSA/SLIK which lacks Spt8 and containing a truncated Spt7 and an additional Rtg2 subunit131. This alternate complex has highly overlapping roles with SAGA, but is linked to the retrograde transport pathway131. Due to 22  the diversity of this module, researchers in the field sometimes split it into the recruitment, architectural, and TBP interaction modules63. For ease of discussion, this thesis will retain the unified SPT module interpretation.  The final module of SAGA is its TAF module consisting of Taf5, Taf6, Taf9, Taf10, and Taf1263. Functional studies of Taf proteins is difficult, as all of them are part of the essential TFIID (Transcription Factor II-D) complex. Taf6, Taf9, Taf10, and Taf12 all contain histone fold domains132. The interconnectedness of these subunits suggest that they function as a scaffold for the SAGA complex.  With a total of two catalytic domains and seven putative chromatin-binding domains, SAGA is a veritable nexus of chromatin modification. The existence of independent complexes resembling the KAT and DUB modules raises the intriguing possibility of independently evolving modules that over time agglomerated into the giant SAGA complex. 1.5.2 Nucleosomal Acetyltransferase of histone H4 (NuA4)  NuA4 is the only essential KAT complex in yeast, and contains thirteen unique subunits for a total molecular weight of approximately 1 MDa23. Akin to SAGA, NuA4 is a transcriptional coactivator that oversees constitutively transcribed housekeeping genes133. Its subunits organize into four distinct modules: the catalytic Piccolo NuA4 module, the Eaf5/7/3 module, the shared SWR1C module, and the recruitment module130,134–136. In addition to the Piccolo module’s role in transcription activation, Esa1 catalytic activity is also required for DNA double strand break repair137. Eaf5/7/3 has been recently implicated in stimulating transcription elongation and recruits NuA4 through interactions with methylated histones45,135. The shared SWR1C module is named thusly due to the fact its component subunits are also found in SWR1C (SWR1 Complex). SWR1C is responsible for integrating the histone variant H2A.Z into 23  nucleosomes138, and the presence of its subunits in NuA4 may link histone acetylation with histone H2A.Z deposition. Interestingly, both NuA4 and SAGA acetylate H2A.Z to further stimulate its deposition139. The recruitment module, which consists of Tra1, functions similarly in NuA4 as it does in SAGA by mediating transcriptional activator recruitment of the complex.  Piccolo NuA4 consists of Esa1, Epl1, Eaf6, and Yng2133,135. This module contains a methylated lysine binding domain: the PHD domain of Yng223. Esa1 contains a Tudor domain that binds poly-U RNA140. Epl1 contains an EPcA domain that is required for Esa1 acetylation activity and mediates the interaction between Esa1 and Yng2134. Piccolo NuA4 also acts separately from NuA4 in maintaining global, activator-independent acetylation levels of histones H2A and H4134. The C-terminus of Epl1, which is separate from its EPcA domain, anchors the module to NuA4134. In addition to its H4 acetyltransferase activity, Piccolo within NuA4 also acetylates nonhistone targets such as Sip2 to repress the activity of its binding partner, the AMP-activated kinase Snf1141. Other nonhistone targets of NuA4 includes Pck1, an essential enzyme for growth on nonfermentable carbon sources142.  The Eaf5/7/3 module has recently been shown to form an active complex independent of NuA4135. This module is enriched over coding regions and mediates the association of NuA4 with phosphorylated RNA polymerase II and methylated histones45,135. Its presence also aids in RNA polymerase II elongation and associated nucleosome destabilization135. Eaf3, which is shared with the RPD3S lysine deacetylase complex, contains a chromodomain and recognizes histone H3K4 and H3K36 methyl marks46. Eaf5 anchors the module to NuA4 through association with the Eaf1 N-terminus143.  Eaf1, which does not associate into a module, serves as the platform for NuA4 assembly. It is the only subunit unique to NuA4 and mediates the association of all four NuA4 modules143. 24  Eaf1 contains a SANT domain, which anchors Tra1 to NuA4, and an HSA (Helicase/SANT-Associated) domain143. The HSA domain mediates the interaction of Act1 and Arp4, both components of the shared SWR1C module, to each other144. Interestingly, Eaf1 is not essential for survival, and suggests that Piccolo NuA4 is sufficient to mediate Esa1’s essential KAT activity143.  The shared SWR1C module consists of Swc4, Yaf9, Act1, and Arp4133. Act1 and Arp4 are an interesting pair of subunits, as Act1 is yeast actin and Arp4 is a very similar protein without ATP (Adenosine TriPhosphate) hydrolysis ability145. Act1 and Arp4 are also found in the Ino80 complex, but their precise functions are yet to be determined. Swc4 contains a SANT domain which may aid in chromatin binding. Yaf9 possesses a YEATS (Yaf9, ENL, AF9, Taf14, and Sas5) domain, a common motif in chromatin modifying complexes, also of indeterminate function146. Interestingly, the human homologue of NuA4—the TIP60 (HIV-1 Tat-Interactive Protein, 60 kDa) complex—greatly resemble a fusion of the NuA4 and SWR1 complexes143.   Similar to SAGA, NuA4 seems to be a merging of multiple modules. The existence of the TIP60 complex in higher eukaryotes raises the intriguing possibility that yeast NuA4 captures a midpoint of convergence, where individual modules construct both SWR1 and NuA4, but the two complexes have not yet fused. 1.5.3 Elongator  The Elongator complex was first discovered associating with the elongating form of RNA Pol II76. Soon after, its Elp3 subunit was identified as a histone and nucleosome acetyltransferase, and mutations in the complex resulted in hypoacetylation of histones H3 and H476,147. Similar to other KAT complexes, mutations in Elongator’s subunits—termed elp mutants—resulted in pleiotropic phenotypes disrupting multiple cellular pathways148. Based on 25  these results, Elongator was considered as a KAT complex that participates in transcriptional pathways overlapping with Gcn5 and SAGA149. However, subsequent research found that yeast Elongator is located in the cytoplasm and did not associate with chromatin in vivo150,151. These observations began to sow doubt into Elongator’s proposed role as a KAT complex. Further evidence mounted against the KAT role of Elongator when the molecular basis of one of elp mutants’ most characteristic phenotypes, resistance to the Kluyveromyces lactis toxin zymocin, was elucidated. The toxin was shown to cleave tRNAs bearing a 5-methoxycarbonylmethyl or 5-carbamoylmethyl modification on its wobble base pair that facilitate codon recognition and enhances translation152,153. Elongator is essential for this modification, and loss of its function removes the zymocin target80. Importantly, overexpression of two tRNAs rescues the pleiotropic phenotypes of elp mutants79. These experiments, coupled with the lack of compelling evidence for chromatin interaction, shifted the consensus from Elongator as a transcription-associated KAT complex to a translational effector instead. Aside from the conflicted opinions of Elongator function, its composition is well-defined. The Elongator complex contains six unique subunits—Elp1, Elp2, Elp3, Elp4, Elp5, and Elp6—and two copies of each subunit associate to form an approximately 850 kDa complex114. Elongator can be further divided into two subcomplexes: Elp123 and Elp456114. The Elp123 subcomplex contains the catalytic Elp3 subunit and mediates the complex’s tRNA modification activity. Elp456 forms a heterohexameric ring also essential for normal Elongator activity154,155. In contrast to SAGA or NuA4, deleting any of Elongator’s subunits result in the same series of pleiotropic elp phenotypes79.  The Elp123 subcomplex is responsible for the complex’s primary catalytic activity. Elp3 contains the radical SAM and GNAT domains to mediate tRNA wobble base pair 26  modification114. Elp1 and Elp2 both contain tandem WD40 domains, and Elp2 has been shown to bind microtubules in vivo and in vitro111,114. The Elp1 C terminus consists of a series of helical TPR repeats and mediates the dimerization of the complex156. Meanwhile, the Elp456 subcomplex dimerizes and forms a six-membered RecA-like ATPase ring154,155. RecA-like ATPases is a large group of ATPases whose members often utilize ATP hydrolysis to power mechanical work157. The Elp456 subcomplex can bind tRNA in an ATP-dependent fashion and may act in a bind-and-release fashion of Elongator’s substrate155. How the two subcomplexes cooperate with each other, and why dimerization is essential for Elongator function remain unclear156. 1.5.4 Mammalian KAT complexes  KAT complexes are conserved in higher eukaryotes where they have diversified to multiple different complexes, serving as a fascinating case study for the evolutionary trajectory of enzyme complexes158. In yeast, two large Gcn5 complexes can be found: SAGA itself and the highly similar SLIK/SALSA131. Humans have three large Gcn5-related complexes: STAGA (Spt-Taf9-Ada-Gcn5 Acetyltransferase), PCAF (P300/CBP-Associated Factor), and ATAC (Ada2A-Containing)158. STAGA has a very similar subunit composition to yeast SAGA and SLIK but contains no homologues for Spt8 or Rtg2159. PCAF resembles a much reduced SAGA, with the Gcn5 homologue PCAF as its catalytic subunit and lacking any deubiquitination module subunits160. ATAC is a particularly interesting KAT, as it contains two catalytic KAT subunits: GCN5 and CSRP2BP161. Aside from the catalytic KAT module, ATAC bears little resemblance to SAGA. In comparison to yeast, human GCN5 has expanded its role to an array of different KAT complexes. 27  Where Gcn5 homologues diversified, the yeast homologue of NuA4, TIP60, consolidated instead. TIP60 contains homologues for nearly all NuA4 and SWR1 complex subunits133. In yeast, the SWR1 complex integrates the histone variant H2A.Z to nucleosomes with various effects in transcription and the DNA damage response162,163. TIP60 takes the mantles of both NuA4 and SWR1, being responsible for histone acetylation and H2A.Z integration164,165. Multiple other KAT complexes only marginally related to yeast KATs arose in humans, such as the MYST-type MSL (Male-Specific Lethal), NSL (Non-Specific Lethal), MOZ/MORF  Table 1.2. Human homologues of the SAGA, NuA4, and Elongator complexes SAGA-related NuA4-related Elongator-like SAGA Sc STAGA Hs PCAF Hs ATAC  Hs SWR1 Sc1 NuA4 Sc Tip60 Hs Elongator Sc Elongator Hs Gcn5 Gcn5 PCAF GCN5/PCAF  Esa1 Tip60 Elp3 ELP3 Ada2 ADA2B ADA2B ADA2A  Yng2 ING3 Elp1 IKBKAP Ada3 ADA3 ADA3 ADA3  Epl1 EPC1/2 Elp2 ELP2 Sgf29 SGF29  SGF29  Eaf6 EAF6 Elp4 ELP4 Tra1 TRRAP TRRAP ZZZ4  Tra1 TRRAP Elp5 ELP5 Spt7 STAF65γ  CRSP2BP Swr12 Eaf12 p400* Elp6 ELP6 Spt3 SPT3 SPT3 HCFC1  Eaf3 MRG15   Spt20 FAM48A  WDR5  Eaf5    Ada1 ADA1  DR1  Eaf7 MRGBP   Spt8   YEATS2 Swc4 Swc4 DMAP1   Taf5 TAF5L TAF5L MBIP Act1 Act1 ACTIN   Taf6 TAF6L TAF6L POLE3 Arp4 Arp4 BAF53   Taf9 TAF9 TAF9 POLE4 Yaf9 Yaf9 GAS41   Taf10 TAF10 TAF10  Bdf1  BRD8   Taf12 TAF12 TAF12  Rvb1  RUVBL1   Ubp8 USP22   Rvb2  RUVBL2   Sgf11 ATXN7L3     MRGX   Sgf73 ATXN7   Vps72  YL1   Sus1 ENY2         Underlined subunits are KAT enzymes. Borders denote conserved modules. Data for this table was compiled from Steunou et al 2014 and Doyon and Côté 2014133,166. 1SWR1 is not a NuA4-related KAT, and is presented here as a comparison to NuA4 and Tip60 2Swr1 is not paralogous to Eaf1, but p400 is homologous to both  28  (MOnocytic leukemia Zinc finger protein/MOZ-Related Factor), and HBO1 (Histone acetyltransferase Binding to ORC1) complexes, as well as the yeast Rtt109-related CBP/p300 (CREB-Binding Protein/p300) complex158.  These KAT complexes play critical roles in humans, and their malfunction carry severe consequences. Their role as regulators of transcription implicates them in many types of cancer. For example, chrosomosomal rearrangements in many cancers result in the fusion of KAT enzymes158. Mutations in CBP/p300 is commonly found in a number of different carcinomas, and amplification of TIP60 subunits has been observed in brain cancers158. Aside from cancer, the polyglutamine expansion of the ATXN7 subunit of SAGA is directly responsible for the neurodegenerative condition spinocerebellar ataxia type 7167,168. This section illustrates that, in addition to being a fundamental cellular process, KAT complexes are important from a human health perspective. 1.6 Techniques for analyzing KAT complexes  A wealth of effort has been dedicated to investigating the structural features of KAT complexes. To date, many catalytic and functional modules found within KATs have been successfully crystallized, providing invaluable molecular insights into their mechanisms of action69,70,84,120,169. However, the structure of the intact complexes remained elusive. SAGA, NuA4, and Elongator contains 19, 13, and 6 unique subunits, respectively. SAGA and NuA4 both contain Tra1, a 400 kDa subunit. These features render recombinant overexpression of these complexes extremely challenging. Even with enough purified protein, crystallizing such large complexes is a monolithic feat on its own. Therefore, alternate techniques will have to be employed to investigate the architecture of intact KAT complexes. 29   In recent years, single particle electron microscopy (EM) has rapidly become the leading technique in investigating large complexes. Single particle EM involves visualizing purified proteins using a transmission electron microscope, either embedded in vitreous ice or a heavy metal negative stain, and compiling data from tens to hundreds of thousands of individual particle images to generate 2D and 3D representations. Since EM does not require crystal formation, the necessary protein concentration is much lower. In recent years, advances in cryo-electron microscopy has resulted in structures of massive >1 MDa complexes at near-atomic resolutions170. However, even low resolution EM analysis using negative stain electron microscopy can reveal important structural features of large multiprotein complexes171–173. Both the power and flexibility of single particle EM makes it an ideal technique for studying KAT complexes.  Although atomic resolution structure determination using EM is possible, practically achieving it remains a very challenging task. In the absence of an atomic resolution EM map or a complete set of X-ray crystallography structures of each subunit, determining the spatial organization of subunits within a complex de novo solely through EM is unfeasible. In recent years, crosslinking coupled to mass spectrometry (CXMS) has proven to be highly complementary to EM analysis of protein complexes174–176. In this experiment, purified protein complexes are chemically crosslinked with a bifunctional crosslinker of a fixed length. The partially crosslinked complex is then enzymatically degraded into peptides and analyzed by liquid chromatography coupled to tandem mass spectrometry177. Specialized algorithms then identify mass signatures that correspond to two crosslinked peptides178. Each crosslinked peptide corresponds to two subunits within a certain distance of each other. Two peptides can therefore only be crosslinked if they are in or near the interface of two subunits. Using these pairwise 30  interactions, the connectivity of the entire complex can be determined. Additionally, CXMS provides distance restraints that can be used to model the subunit spatial organization even in low resolution EM structures.  The combination of single particle EM and CXMS provides valuable information on the molecular architecture of complexes. Information gleaned from these experiments can formulate many hypotheses to guide biochemical and genetic experiments while the significant challenges of high resolution structure determination are being addressed. 1.7 Objectives of thesis  Regulation of gene expression is a fundamental requirement for all complex life. The human body can adapt to an amazing number of conditions such as temperature extremes, nutrient deprivation, oxygen shortage, and pathogenic insults. Many of our most basic adaptation mechanisms stem from the ability of our cells to change their transcriptional regime to compensate for environmental challenges. Conversely, misregulation of gene expression leads to the leading cause of death in Canada: cancer (Statistics Canada 2011). Histone acetylation is a central regulator of transcription, and histone deacetylase inhibitors are a promising avenue for cancer therapy. Despite the importance of histone acetylation, the field knows mostly fragments about the molecular mechanisms driving the effectors of this epigenetic mark. How the modules and subunits of the SAGA, NuA4, and Elongator lysine acetyltransferase complexes spatially organize remains an open question. This dissertation aims to provide a detailed structural model of the SAGA, NuA4, and Elongator complexes as the first step towards understanding how multiple different functionalities cooperate within these enigmatic complexes.  Chapter 2 describes the characterization of the structure of the intact yeast SAGA complex. Data obtained through single particle electron microscopy and crosslinking coupled to 31  mass spectrometry were combined to provide a model of SAGA subunit organization. This work is published in the Journal of Biological Chemistry179.   Chapter 3 describes the characterization of the yeast NuA4 complex and initial structural investigations into the human homologue of NuA4: TIP60. This chapter describes the optimization of NuA4 purification and subunit organization determined by single particle electron microscopy and crosslinking coupled to mass spectrometry.   Chapter 4 describes the characterization of the yeast Elongator complex and presents the first look into its intact structure. This chapter presents single particle electron microscopy, crosslinking coupled to mass spectrometry, and mutational analysis to present a detailed model of Elongator molecular architecture. This work is published in EMBO Reports180.         32  Chapter 2: Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 Acetyltransferase complex 2.1 Introduction The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex is a highly conserved histone acetyltransferase (HAT) complex that activates the transcription of stress response genes in yeast63. As the largest HAT complex, SAGA consists of 19 core subunits that associate into a stable assembly of approximately 1.8 MegaDaltons (MDa) in overall mass, with Gcn5 serving as the catalytic subunit for acetylating histone H3 (Table 2.1)63. Several studies have sought to elucidate the structural features of SAGA. Single-particle electron microscopy (EM) analysis by Wu et al provided the first step towards understanding the overall structure of this complex by generating the first three-dimensional (3D) reconstruction of SAGA, and localizing nine core subunits using antibody labeling techniques181. Two-dimensional (2D) analysis in this study also revealed that a sub-population of SAGA particles possesses an additional region of density, which can adopt different conformations. A recent study mapping the DUB module to an EM structure of SAGA also observed this sub-population182. More recently, an EM study on human TFIID showed that this complex undergoes massive rearrangement that alters both the position and the connectivity of an entire lobe183. Even though SAGA shares multiple core subunits with TFIID, whether the observed structural flexibility is a shared property of these complexes is not known. Furthermore, at the time of the first EM study, SAGA’s H2B deubiquitination activity and the subunits associated with this catalytic activity have not been identified56. Finally, apart from the DUB module, there is a dearth of high resolution structural information for the other SAGA subunits, rendering our structural understanding of the complex incomplete.   33  Table 2.1. Subunits of yeast SAGA Module   Subunit MW (kDa) HAT module Ada21 50.6   Ada3 79.3   Gcn52 51.1   Sgf29 29.4 DUB module Sgf11 11.3   Sgf731 72.9   Sus1 11.1   Ubp82 53.6 Spt module Ada1 54.5   Spt3 38.8   Spt7 152.6   Spt8 66.2   Spt20 67.8   Tra1 433.2 Taf module Taf5 89.0   Taf6 57.9   Taf9 17.3   Taf10 23.0   Taf12 61.1 1Anchors module to complex   2Catalytic subunit     By developing a modified purification strategy that enhances the stability of SAGA, we uncovered the remarkable conformational flexibility of this complex using single-particle electron microscopy (EM). Systematic subunit deletions and mutations enabled us to further dissect the role of the different modules in mediating structural rearrangement. By combining established EM-based labeling methods with chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS), we mapped and validated the spatial location of all core components of SAGA, including subunits of the DUB module. Collectively, our data enabled us to generate a model for SAGA’s molecular organization and to gain insights into the physiological relevance of its conformational flexibility. 34  2.2 Experimental methods 2.2.1 S. cerevisiae strain construction Haploid strains BY4741, BJ2168, and SF1 backgrounds were used to construct all the strains in this study (Table 2.2). TAP- (Tandem Affinity Purification) and GFP (Green Fluorescent Protein)-tagged strains were constructed using PCR amplified inserts from plasmids pBS1539 and pFA6a-GFP(S65T)-kanMX6, respectively. These inserts contain the TAP- and GFP-tags as well as a selection marker flanked by approximately 40 bp sequences homologous to the genomic sequence just 5’ and 3’ of the target protein’s stop codon184,185. The inserts were then transformed into yeast using lithium acetate, polyethylene glycol, and single-stranded carrier DNA incubation and heat shock186. Deletion strains were similarly constructed using PCR amplified inserts from pFA6a-kanMX6185. FLAG-tagged strains were also constructed using PCR amplified inserts from pMK144-3xFLAG-kanMX6. Proper integration of inserts was checked by colony PCR187, and expression of tags was evaluated using Western Blot with α-TAP antibody (Thermo Scientific), α-GFP antibody (Roche), or α-FLAG M2 antibody (Sigma). Standard methods for S. cerevisiae culturing were used.  2.2.2 Purification of native SAGA Native SAGA was purified by a traditional TAP purification181,184, or a modified version substituting the calmodulin binding step with gradient fixation (GraFix)188. In particular, TAP-tagged yeast cells were grown to an OD600 of ~2.5, harvested by centrifugation, and the cell pellets frozen at -80°C. 20 to 25 g of frozen cells were resuspended in ~80 ml lysis buffer (40 mM HEPES pH7.4, 350 mM NaCl, 10% glycerol, 0.1% Tween-20, 1 mM PMSF, 50 mM NaF, 0.1 mM Na3VO4, 2 mM benzamidine, and EDTA-free protease inhibitor (Roche) and lysed by bead beating. The lysate was then centrifuged at 30,000x g for 30 minutes. Clarified lysates were  35  Table 2.2. S. cerevisiae strains used in this study of SAGA Strain name Genotype SF1 MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1 ura3-1 SF1 Spt7-TAP MATa SPT7-TAP::URA3 ade2-1 can1-100 his3-11 leu2-3,112 trp1 ura3-1 BJ2168 MATa leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP MATa SPT7-TAP::URA3 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-3xFLAG MATa SPT7-FLAG::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Δada2 MATa ada2::KanMX6 SPT7-TAP::URA3 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Δsgf73 MATa sgf73::KanMX6 SPT7-TAP::URA3 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Gcn5-GFP MATa SPT7-TAP::URA3 GCN5-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Ada2-GFP MATa SPT7-TAP::URA3 ADA2-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Sgf29-GFP MATa SPT7-TAP::URA3 SGF29-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Spt3-GFP MATa SPT7-TAP::URA3 SPT3-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Spt8-GFP MATa SPT7-TAP::URA3 SPT8-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Spt20-GFP MATa SPT7-TAP::URA3 SPT20-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Taf5-GFP MATa SPT7-TAP::URA3 TAF5-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Taf9-GFP MATa SPT7-TAP::URA3 TAF9-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BJ2168 Spt7-TAP Taf10-GFP MATa SPT&-TAP::URA3 TAF10-GFP::KanMX6 leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2 BY4741 MATa his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Sgf73-TAP MATa SGF73-TAP::URA3 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Sgf73-TAP Spt7(1-1180) MATa SGF73-TAP::URA3 spt7-1180::KanMX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Gcn5-GFP MATa GCN5-GFP::KanMX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Gcn5-GFP Δada2 MATa GCN5-GFP::KanMX6 ada2::HIS3MX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 36  Table 2.2. S. cerevisiae strains used in this study of SAGA (cont.) Strain name Genotype BY4741 Gcn5-GFP Δsgf73 MATa GCN5-GFP::KanMX6 sgf73::HIS3MX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Ubp8-GFP MATa UBP8-GFP::KanMX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Ubp8-GFP Δada2 MATa UBP8-GFP::KanMX6 ada2::HIS3MX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Ubp8-GFP Δsgf73 MATa UBP8-GFP::KanMX6 sgf732::HIS3MX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 BY4741 Ubp8-GFP Δsgf73 SGF73 MATa UBP8-GFP::KanMX6 sgf732::HIS3MX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 pRS316-SGF73-FLAG (pYH28121) BY4741 Ubp8-GFP Δsgf73 sgf73(2-104Δ) MATa UBP8-GFP::KanMX6 sgf732::HIS3MX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 pRS316-sgf73-(2-104Δ)-FLAG (pYH67121) BY4741 Ubp8-GFP Δsgf73 sgf73(350-400Δ) MATa UBP8-GFP::KanMX6 sgf732::HIS3MX6 his3Δ1 leu2Δ20 met15Δ0 ura3Δ0 pRS316-sgf73-(350-400Δ)-FLAG (pYH39121)  incubated with 500µl IgG Sepharose (GE Healthcare) at 4°C for 1.5 hours. The IgG resin was washed with IPP150 buffer (40 mM HEPES pH7.4, 150 mM NaCl, 0.2% NP-40, and 10% glycerol) and resuspended in 750 µl TEV-C buffer (40 mM HEPES pH7.4, 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA, 10% glycerol, and 1 mM DTT). Bound proteins were eluted by tobacco etch virus (TEV) protease cleavage at 16°C for 1.5 hours. 2 µl of 10 mg/ml RNAse A was added to 500 µl of the eluate and incubated on ice for 30 minutes. 2 x 200 µl of the eluate were overlaid on two linear 15-30% glycerol gradients, with one containing 0.00-0.05% glutaraldehyde cross-linker prepared using the Gradient Station (BioComp). The gradients were spun at 58,800x g for 16.5 hours at 4°C. The gradients were then fractionated using the Gradient Station. Fractions from gradients without cross-linker were TCA precipitated for silver stain SDS-PAGE analysis, and corresponding fractions with cross-linker, concentrated using 100K centrifugal filter units (Millipore) if necessary, were used for further EM analysis. 37  For antibody binding and cross-linking mass spectrometry analyses, SAGA was purified using anti-FLAG affinity chromatography. 3xFLAG-tagged Spt7 yeast cell pellets were obtained as before. Frozen pellets are pooled and ground using a freezer mill (SPEX SamplePrep 6870) under liquid nitrogen temperatures. 35 to 40 ml of the finely ground cell lysate was resuspended in ~80 ml lysis buffer. The lysate was centrifuged at 30,000x g for 30 minutes. Clarified lysates were incubated with 500 µl α-FLAG M2 resin (Sigma) for 1 hour. The resin was washed with 3x5 ml lysis buffer without inhibitors, and 1x5 ml reduced salt (150mM) lysis buffer without inhibitors. The resin was resuspended in 1 ml reduced salt lysis buffer containing 2.5 µg/ml RNAse A, and incubated at 4°C for 30 minutes. The resin was washed with 2x5ml reduced salt lysis buffer. Bound proteins were eluted twice in 500µl of reduced salt lysis buffer without inhibitors and 0.5 mg/ml 3xFLAG peptides.  2.2.3 Negative stain sample preparation and imaging Negatively stained specimens for 2D analysis were prepared from purified SAGA as described previously171. 3 μL of ~10 μg/ml sample was applied to copper grids (400 mesh copper, Ted Pella G400) prepared by layering with amyl acetate and carbon coating with a carbon evaporator (108C Auto Carbon Coater, Ted Pella). Samples were adsorbed for 30 seconds to 4 minutes as needed, blotted off on Whatman 4 filter paper (Fisher Scientific), then washed twice in water and once in 0.7% uranyl formate solution. Grids were then stained for 30 seconds in 0.7% uranyl formate and blotted off. Samples for 3D analysis were prepared using the carbon sandwich negative staining technique to improve stain embedding and to minimize sample flattening171. Similar procedures were used for sandwich negative staining as regular negative staining, except the final staining step: a small square of carbon prepared on mica strips (Ted Pella) is floated on a pool of 0.7% uranyl formate solution, and the sample-adsorbed grid is 38  slid into the stain and lifted out such that the carbon overlays onto the its surface. Excess stain is blotted with a strip of filter paper.  Samples were visualized using a Tecnai Spirit transmission electron microscope (FEI) operated at an accelerating voltage of 120kV. Images were taken at a nominal magnification of 49,000x using an FEI Eagle 4K x 4K charge-coupled device (CCD) camera at a defocus value of -1.2 µm under low dose conditions. For tilt pair data collection, the same parameters were used and two images, one at 60° tilt and one untilted, were taken from the same specimen area. 2 x 2 image pixels were then averaged for a final pixel size of 4.7 Å. 2.2.4 Image processing For 2D analysis, individual particle images were interactively selected using Boxer189. The selected particles were then windowed into 128 x 128-pixel images, rotationally and translationally aligned, and subjected to K-means classification to generate class averages using the SPIDER image processing suite190. For GFP-tagging analysis, we used a preliminary round of reference-free classification and averaging to visualize regions with possible additional density. These averages were used to create masks that focused on the vicinity of the additional density, and the areas within the masks were used to re-classify the input particle images. Averages obtained from this method were compared to untagged SAGA of a similar conformation via subtraction analysis. Images of untagged SAGA were subtracted from images of tagged SAGA, and the resulting difference image was threshold to find signals that were either 3 or 4 standard deviations from the mean pixel value.  For determining the de novo 3D reconstructions of SAGA, 22,518 pairs of particle images were first selected using WEB190. Particles in the untilted set were windowed into 128 x 128-pixel images, aligned, and classified into 50 classes using SPIDER. Two class averages that 39  correspond to each of the three conformations were merged. 3D reconstructions were generated from the tilted particles of these combined averages using the backprojection and angular refinement algorithms in SPIDER. The final resolutions of the 3D models were estimated by the Fourier shell correlation (FSC) function using the 0.5 FSC criterion. Due to the flexibility of the complex, we chose to report the more conservative 0.5 FSC criterion for resolution estimation as opposed to the gold standard 0.143 cutoff. The curved, arched, and donut reconstructions had resolutions of 42 Å, 45 Å, and 39 Å, respectively. Molecular docking analysis and model construction was performed using UCSF Chimera191. 2.2.5 Conformation population analysis Conformation population analysis similar to a previous study was conducted192. Particle measurements were done using ImageJ193. 100 class averages from the Spt7-TAP wild-type and sgf73 strains were used. Only averages with unambiguous outlines corresponding to SAGA were analyzed. The length of the cleft formed in the arched conformation of SAGA and the shortest distance between the distal end of the tail and the shoulder were measured. Three independent measurements were made, and the averages used to create combined scatter and bar plots.  2.2.6 Antibody labeling of SAGA Non-cross-linked SAGA purified from Sgf73-TAP-tagged strain was incubated with 10 µg/ml α-TAP antibody (Thermo Scientific) at room temperature for 10 minutes and used for single particle EM analysis. Particles with bound antibodies were selected for 2D analysis as detailed above. 2.2.7 Chemical cross-linking and mass spectrometry analysis FLAG-purified SAGA was concentrated from 2 ml to ~100 µl using 100K centrifugal filters (Millipore) and were cross-linked with DSS as described194, precipitated with 4 volumes 40  of cold acetone, washed once with cold acetone, air-dried, and then dissolved in 8 M Urea, 100 mM Tris, pH 8.5. After 5 mM TCEP reduction and 10 mM IAA alkylation, the samples were digested with Lys-C at a 1:100 enzyme:substrate ratio at 37 °C for 4 h. The samples were 4-fold diluted with 100 mM Tris, pH 8.5 before they were digested with trypsin (1:50, enzyme:substrate) at 37°C. After 12 h, formic acid (FA) was added to a final concentration of 5% to stop digestion. The samples were cleared by centrifugation at 14,000 rpm for 10 min and desalted with a home-made 250 µm x 1 cm C18 reverse phase column. Desalted peptides were loaded onto a 75 µm x 10 cm analytical column packed with 1.8 µm, 120 Å UHPLC-XB-C18 resin (Welch Materials Inc.) and separated over a 107-min linear gradient from 100% buffer A (0.1% FA) to 30% buffer B 100% acetonitrile, 0.1% FA), then a 10-min gradient from 30% to 80% buffer B, and maintaining at 80% buffer B for 6 min before returning to 100% buffer A in 5 min and ending with a 9-min 100% buffer A wash. The flow rate was 200 nL/min. The Easy-nLC 1000 UPLC was coupled to a Q Exactive mass spectrometer (Thermo-Fisher Scientific). The MS parameters were: Top 20 most intense ions in a survey full scan were selected for MS2 by HCD dissociation; R = 140,000 in full scan, R = 17,500 in HCD scan; AGC targets were 1e6 for FTMS full scan, 5e4 for MS2; minimal signal threshold for MS2 = 4e4; precursors of charge states +1, +2, > +8, or unassigned were excluded; normalized collision energy = 30 for HCD; peptide match is preferred. 2.2.8 Expression and purification of recombinant transcriptional activators Coding regions of Gcn4, TBP, and Gal4 activation domain (residues 768-881) were PCR amplified from yeast genomic DNA 195. The PCR products were cloned into the NdeI/EcoRI sites of pET28b-HMT vector. BL21* (Life Technologies) E. coli expression strain transformed with the resulting constructs were grown to an OD600 of 0.5 and induced with 1mM IPTG for 41  either 3 hours at 37°C or overnight at 16°C. The bacteria were then harvested by centrifugation and stored at -80°C. For each purification, a frozen cell pellet was resuspended 10 mL/g in lysis buffer (40 mM HEPES pH 8.0, 500 mM NaCl, and 2 mM PMSF). The cells were lysed by sonication, and the lysate was clarified by centrifugation at 30,000x g. The clarified lysates were then incubated with 500 µl of Ni-NTA Sepharose (Thermo Scientific) for 30 minutes at 4°C. The resin was washed with 3x5 ml lysis buffer, and then 2x5 ml lysis buffer with 50mM imidazole. Bound proteins were eluted in 5 rounds, using 1 ml of lysis buffer with 250 mM imidazole. Fractions containing the desired protein were concentrated using a 10K centrifugal filter (Millipore) and further purified by gel filtration chromatography (GE Healthcare). Peak fractions containing pure activator proteins were flash frozen in liquid nitrogen and stored at -80°C. 2.2.9 Activator pulldown experiments and Western blotting Approximately 50 ng/µl of FLAG-purified SAGA was mixed with 300 µg/ml HMP (His-Maltose Binding Protein) and HMP-tagged Gcn4, Gal4AD (Gal4 Activation Domain), and TBP in binding buffer (40 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Tween-20, 0.5 mM DTT, and 1 mM PMSF). The mixture was incubated with 50 µl amylose resin (NEB) for 30 minutes at 4°C. The resin was collected using centrifugal spin columns and washed twice with 200 µl binding buffer. Bound proteins were eluted with 200 µl binding buffer with 100 mM maltose. Eluates were then analyzed by SDS-PAGE and Western Blot using the following antibodies: mouse α-FLAG antibody (Sigma), mouse α-His antibody (ABM), and α-mouse HRP antibody (Sigma).   42  2.2.10 Activator binding localization experiments FLAG and glycerol gradient-purified SAGA was mixed with 4-8 µg/ml of purified activators and incubated at room temperature for 30 minutes before negative-stained EM specimens were prepared. 2.2.11 Modeling  Modeling of subunit structures and spheres approximating subunits were conducted in UCSF Chimera191. Subunits are manually placed according to EM labeling experiments and previous knowledge of the structural organization of modules. Distance restraints from CXMS experiments were visualized using the pseudobond feature of Chimera, and the organization further refined to minimize the distance between crosslinked subunits. Due to the lack of high resolution structure or homology models of SAGA subunits, spheres approximating the volume of each subunit were used as placeholders. The inherent imprecision of using spheres mixed with high resolution structures preclude energy minimization approaches for modeling SAGA architecture. 2.2.12 SAGA module localization  BY4741 yeast strains were used for SAGA module localization (Table 2.2). Indicated subunits were genomically tagged using C-terminal eGFP, or replaced through homologous recombination with auxotrophic markers. Several strains were transformed with pRS316-based vectors carrying complementing genes as indicated. Overnight cultures were used to inoculate synthetic media designed to maintain auxotrophic plasmid maintenance at an initial OD600 of 0.5 and grown at 30ºC for 3 hours. 1 mL of culture is centrifuged at 14,000 rpm, and the pellet is used to prepare slides without fixing. Cell pellets were diluted using the same culture medium. 43  Slides were visualized using the Olympus FluoView FV1000 confocal microscope (Olympus) using the eGFP filter set. 2.3 Results 2.3.1 Improved procedure for isolating native S. cerevisiae SAGA The tandem affinity purification (TAP) procedure is an established method for isolating native SAGA from yeast containing genomically tagged SPT7-TAP181,184. We combined parts of TAP with the gradient fixation (GraFix) technique, which involves subjecting the complex to limited glutaraldehyde cross-linking during glycerol gradient ultracentrifugation196. GraFix has been shown to maintain complex integrity and increase EM image quality188. To ensure that SAGA is amenable to glycerol gradient ultracentrifugation, we analyzed fractions from a corresponding glycerol gradient lacking glutaraldehyde by SDS-PAGE. SAGA migrates to a single fraction with minimal contaminants (Fig 2.1A). Subsequent mass spectrometry analysis confirmed that this fraction contains all 19 core SAGA subunits (Table 2.3).  We next examined the purified SAGA using negative stain electron microscopy. We observed particles of similar size and shape as those observed by Wu et al from samples purified by conventional 2-step TAP method and by non-cross-linked glycerol gradient ultracentrifugation. However, the samples from the TAP purification and glycerol gradient purification showed a high degree of subunit dissociation (Fig 2.1B). Meanwhile, samples that underwent GraFix treatment display not only significantly reduced the level of sample heterogeneity but also preserved fine structural features of individual SAGA particles (Fig 2.1B).  2.3.2 SAGA adopts three distinct conformations To gain further insights into the structural properties of SAGA, we applied a two-dimensional (2D) single-particle EM approach that involves classifying manually selected  44   Figure 2.1. Purification of S. cerevisiae SAGA and negative stain EM analysis. (A) SDS-PAGE analysis of yeast SAGA purified from IgG Sepharose and 15-30% glycerol gradient ultracentrifugation. Protein bands were visualized by silver-staining, and the fraction marked with the arrow was used for EM and mass spectrometric analysis (Table 2.3).  (B) A representative raw image of negatively-stained TAP-purified or GraFix-purified SAGA. (C) Representative 2D class averages of SAGA purified by TAP, 45  glycerol gradient ultracentrifugation without cross-linker, and GraFix. The three GraFix class averages correspond to the three observed conformations. Side length of every class average panel is 60 nm.  Table 2.3. Mass spectrometry analysis of purified SAGA unit composition   WT  ada2Δ  sgf73Δ Subunit MW (kDa) Peptide  # Coverage (%)  Peptide # Coverage (%)  Peptide # Coverage (%) Tra1 432.9 59 21.1  23 7.7  18 6.5 Spt7 152.5 37 35.4  17 17.3  11 12.1 Taf5 88.9 38 59  30 49.9  33 49.8 Ada3 79.2 30 52.6  0 N/A  19 42.9 Sgf73 72.8 20 44.4  16 39  0 N/A Spt20 67.8 26 58  26 59.9  30 66.9 Spt8 66.1 21 46.7  20 47.5  18 39.9 Taf12 61 23 53.6  20 42.9  23 49.5 Taf6 57.9 19 51  18 46.5  22 53.3 Ada1 54.4 19 48.4  17 46.1  21 55.9 Ubp8 53.6 23 55  15 35.5  1 2.9 Gcn5 51 21 60.8  2 5.7  19 51.7 Ada2 50.5 19 57.6  0 N/A  17 57.1 Spt3 38.8 9 30.9  7 24.6  11 35.6 Sgf29 29.4 10 47.1  2 10.4  10 46.7 Taf10 23 8 63.1  5 47.1  7 49 Taf9 17.3 4 36.9  4 39.5  4 42.7 Sgf11 11.3 1 17.2  1 17.2  0 N/A Sus1 11.1 2 32.3  4 64.6  0 N/A  particles according to similarities in overall morphology, and aligning and calculating an average image of the particles constituting each class. The class averages of TAP purified and glycerol gradient purified SAGA without cross-linker were practically identical to each other and to previously published images (Fig 2.1C)181. However, class averages of SAGA purified using GraFix showed improved image quality and better-resolved features (Fig 2.1C). These class 46  averages, calculated from 7,753 GraFix-purified particles, showed that SAGA consists of a prominent globular ‘head’ and a long and slender ‘tail’ separated by a ‘torso’ region. The torso region can be further subdivided into two halves: a ‘joint’ that is connected to the tail, and the ‘shoulder’ that does not make a direct connection with the tail. Interestingly, the prominent extended tail that we observed is only found in a small population of particles in the previous EM analysis of SAGA, while we observed the tail in 91% of our particles. We attributed this discrepancy to the fact that the tail portion of SAGA is more labile and has a tendency to dissociate from the complex upon purification and/or during the negative staining specimen preparation procedure.  Most strikingly, our analysis revealed that the head, torso, and tail regions of SAGA are all conformationally flexible. In particular, the tail region can curve and sample a broad range of space. The coordinated movement of multiple densities within SAGA is remarkable due to large distances covered; the tip of the SAGA tail traverses over 50 Å between different conformations. From the gallery of class averages, three major types of conformations could be distinguished based on the arrangement of the mobile tail region with respect to the rest of the complex (Fig 2.1C). In the ‘donut’ conformation, the tail curls up with its tip pointing towards the shoulder of the torso to generate an almost complete circular structure at the bottom half of the complex. In the ‘arched’ conformation, the tail retracts from the shoulder to generate a kink and a pronounced deep cleft at the back of the torso. In the ‘curved’ conformation, the tail adopts a gentle curvature with its tip projected away from the shoulder. Interestingly, the different tail arrangements are accompanied by changes in the morphology of the SAGA head and shoulder regions. We observed multiple class averages that would fall into intermediate states among the three major conformations. Collectively, our 2D analysis suggests that SAGA is structurally dynamic and its 47  conformational changes involve coordinated movements and rearrangements of different subunits and modules within the complex.  2.3.3 Removal of key subunits affects SAGA’s conformational flexibility We next examined the effects of subunit and module deletions on SAGA’s conformational plasticity. Previous studies have shown that deleting the ADA2 gene dislodges the HAT module from SAGA while leaving the rest of the complex intact116. Our mass spectrometry analysis confirmed this finding by showing that subunits of the HAT module are absent in SAGA isolated from the ada2Δ yeast strain (Table 2.3). Subsequent EM analysis of SAGA devoid of Ada2 revealed that the tail region is severely shortened in all class averages, suggesting that the HAT module likely constitutes a distal segment of the tail (Fig 2.2B). In spite of the reduced size of the tail region, the absence of the HAT module does not diminish SAGA’s conformational flexibility. In fact, the shoulder region of this mutant SAGA shows even greater mobility, translocating away from the head and towards the tail. Thus, while the HAT module does not influence SAGA’s ability to adopt different conformations, its absence induces additional structural variability in other regions of SAGA.  SALSA/SLIK is an alternate form of yeast SAGA that lacks the Spt8 subunit due to a C-terminal truncation of the Spt7 subunit197. To emulate SALSA/SLIK, we generated a yeast strain containing a truncation of the SPT7 gene from residue 1180 to the C-terminus. Negative stain 2D EM revealed that this SALSA/SLIK complex lacks the shoulder region, but the mutation does not affect the conformational flexibility of the tail (Fig 2.2C). Spt8 likely comprises a significant portion of the shoulder region, and is a peripheral subunit as its absence does not dramatically alter the rest of this complex.  48   Figure 2.2. Effects of mutations on SAGA flexibility.  SAGA containing subunit deletions were analyzed using single particle EM for loss of density and changes in flexibility. (A-C). Class averages from wild-type, ada2Δ, and truncated spt7(1-1080) SAGA arranged to display their range of flexibility. Yellow circles show electron 49  densities that are absent from the mutants compared to wild-type. Schematics of SAGA demonstrates the movement of regions and missing densities within the complex. (D-E) Conformational population analysis of wild-type and sgf73Δ SAGA. Description of the measurements used are shown in D. Bar graphs represent the total number of particles measured for each value on the corresponding axis.   Deletion of the SGF73 gene has been previously shown to dissociate the DUB module from SAGA116. Although we confirmed that SAGA isolated from the sgf73Δ strain lacks the  DUB module, the 2D class averages of this mutant complex show no apparent loss of density (Fig 2.2D, Table 2.3). We did observe an increased number of particles with the HAT module absent, suggesting that SGF73 deletion modestly destabilizes the complex. A centrally located DUB module would render the loss of density more difficult to observe, explaining our observation. Intriguingly, the sgf73Δ SAGA mutant appears to have a lower propensity than wild type SAGA to adopt the donut conformation. To more accurately assess the shift in occupancy of the different conformation states between wild type SAGA and the sgf73Δ mutant complex, we defined the three major conformations based on two measured parameters: the shoulder-to-tail distance, and the cleft length (Fig 2.2D). More specifically, we defined the donut conformation to have a shoulder-to-tail distance under 20 Å and the arched conformation to have a cleft length greater than 15 Å, with precedence given to the former. Based on this analysis, we found that 28% of wild-type SAGA adopted the donut conformation, compared to only 4% by the sgf73Δ mutant complex. The DUB module is therefore necessary for SAGA to efficiently adopt the donut conformation, either by stabilizing the rearrangements involved in the movement of the tail, or physically mediating the connection between the 50  shoulder and the tail. Our results show that different SAGA modules contribute to the complex’s flexibility in varying degrees.  2.3.4 3D reconstructions of SAGA in three conformations SAGA’s intrinsic heterogeneity and the low yields from our endogenous purification procedure precluded high-resolution cryo-EM analysis. Instead, we generated de novo 3D reconstructions of SAGA using the random conical tilt approach198. This approach was chosen because SAGA adopts a preferred orientation on the carbon support layer of the EM grids, precluding the use of common line techniques that require comprehensive angular representation. We selected tilted particles corresponding to the curved, arched, and donut conformations, and used them to calculate 3D reconstructions. Despite the intrinsic flexibility of the complex, we were able to visualize the structural rearrangements associated with the conformational shifts (Fig 2.3A-C). Notably, we observed that the tail undergoes a large degree of rearrangement between the three conformations. The arched conformation showed disconnected density in the middle of the tail, an observation that is indicative of a particularly heterogeneous region. Furthermore, the shoulder and its adjacent head region also displayed substantial rearrangements, with multiple shifting densities. In the donut conformation, the shoulder region splits into two separate densities and shifted away from each other. In spite of its limiting resolution, our 3D analysis demonstrated that transition between the three conformations require large-scale structural rearrangement of the subunits within the complex. A recent study by Durand et al also generated a 3D reconstruction of SAGA purified without the use of GraFix182. The overall configuration of Durand et al’s map corresponds most closely to our SAGA donut reconstruction (Fig. 2.3D), although the precise distribution of densities vary slightly between the two. The   51   Figure 2.3. Three-dimensional reconstructions of the three SAGA conformations. (A-C). Models were generated de novo from both tilted and untilted particles that correspond to each SAGA conformation using the random conical tilt method. (D) 3D reconstruction of SAGA purified without gradient fixation by Durand et al (EMDB code: 2693). (E) The mTOR C-terminal crystal structure (PDB code: 4JSV199), which shares a high degree of secondary structure similarity with the Tra1 C-terminus, does not fit within the region of SAGA’s head previously proposed to contain Tra1. (F) The region that likely contains the entire Tra1 structure is highlighted in green.  52  different crosslinking method employed by their study may cause different conformations to be stabilized, resulting in the dissimilarities between the reconstructions. 2.3.5 Tra1 occupies a substantial portion of SAGA’s head region Upon examination of the SAGA 3D reconstructions, we sought to evaluate Wu et al’s proposal that the Tra1 subunit resides within one half of the head region. Further evidence of this localization comes from the NuA4 HAT complex EM structure, which bears a striking resemblance to the head region of SAGA, while sharing only the Tra1 subunit200. At 400kDa, Tra1 is the largest subunit of SAGA and is thought to be responsible for recruitment of this complex to its target genes117. Tra1 is a pseudokinase that belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) family of extraordinarily large protein kinases, whose members share a common domain organization: extensive tandem HEAT repeats at its N-terminal region, followed by the FAT (FRAP-ATM-TRRAP), kinase, and FATC (C-terminal FAT) domains at the C-terminal region130. Although there is no high-resolution structural data available for Tra1, the crystal structure of the 1,174 C-terminal residues of mTOR (mammalian Target Of Rapamycin), a PIKK protein which shares a high degree of secondary structure similarity to the predicted C-terminal domain of Tra1, has been reported201. We used this crystal structure to evaluate the proposed location of Tra1. We found that even at less than one-third the size of full length Tra1, the mTOR C-terminal domain was too large to fit within the region proposed by Wu et al (Fig. 2.3D). Furthermore, a low-resolution crystal structure of another PIKK protein, DNA-PKcs, shows a globular region with slender projections that form a ring202, reminiscent to the arrangement of electron density within the SAGA head. Although our 3D reconstructions are of insufficient resolution for further computational docking analysis, we believe that based on size 53  alone these comparisons demonstrate that Tra1 likely occupies a large proportion of both lobes of the prominent head region (Fig 2.3E).  2.3.6 Comprehensive EM-based mapping of subunit localization Using an antibody-based approach, Wu et al has deduced the positions of several subunits within SAGA. To validate these subunit locations in light of our ability to visualize SAGA with a fully extended tail and to further expand this analysis, we applied a proven labeling approach that involves introducing C-terminal green fluorescent protein (GFP) tags to different SAGA subunits203. We purified the corresponding GFP-tagged SAGA complexes, and located the additional electron density introduced by GFP by negative stain 2D EM methods.  Our earlier ADA2 deletion experiment suggested that the HAT module makes up the tail region of SAGA. In agreement with this result, SAGA containing GFP-tagged Ada2, Gcn5, or Sgf29 all display additional density centered about the tail region (Fig 2.4A). Our analysis showed that Gcn5 localizes to the tip of the tail region, whereas Wu et al localized this subunit within the shoulder region181. This discrepancy may be explained by the instability and potential dissociation of the tail region of non-cross-linked SAGA. The localization of SAGA’s HAT activity to the most mobile region of the complex provides a tantalizing explanation for its ability to act on a wide range of chromatin templates.  We next investigated the localization of the TAF module subunits Taf5, Taf9, and Taf10. SAGA containing GFP-tag fused to these three subunits resulted in additional electron densities observed near the torso joint region (Fig 2.4B). Although SAGA is thought to contain two copies of Taf5, Taf6, Taf9, and Taf12, we did not observe two unambiguous densities for GFP-tagged Taf5 and Taf9181. This is likely due to a central location of the second copy of each protein, where other protein density can obstruct the visualization of the second GFP density. The TAF 54   Figure 2.4. Localization of SAGA domains and subunits. (A-C). Class averages of untagged wild-type (WT) SAGA and SAGA containing GFP-tagged subunits were compared to determine the approximate locations of the tagged subunits. White arrows denote the additional density on the GFP-tagged image. The third images of each set are difference maps between tagged and untagged SAGA, thresholded to show only signals 3 or 4 standard deviations (σ) above the average pixel value. (D) To localize the DUB module, SAGA purified using Sgf73-TAP subunit was incubated with α-TAP 55  antibody and visualized using single particle EM. Yellow circles denote density attributed to the antibody. (E) Proposed locations of the modules shown on the schematic of SAGA.  module therefore resides within the torso region of SAGA. Since SAGA containing a truncated tail still displays extensive flexibility, the central location of the TAF module supports our proposal that the shared TFIID subunits mediate a large degree of the complex’s conformational changes. We next targeted the Spt3, Spt8, and Spt20 subunits of the SPT module. Consistent with our truncated Spt7 findings, the corresponding GFP-tagged SAGA complexes all displayed an additional density near the shoulder region and central torso of SAGA (Fig 2.4C). The flexibility of the shoulder region, where both Spt3 and Spt8 reside, may be necessary to accommodate TBP binding and release from these subunits. Based on the size and number of the SPT module subunits, some of these proteins likely occupy the torso region near the TAF module subunits. Previous EM studies placed Spt20 on the opposite end of SAGA from Tra1181, a location that disagrees with our central localization. However, our observation is consistent with Lee et al’s subunit depletion study that proposed Tra1 and Spt20 to be in close proximity to each other116.  When we applied the same experimental approach to map the location of subunits of the DUB module, we were unable to find any additional density clearly attributable to GFP. Furthermore, we were also unable to unambiguously fit the published crystal structure of the DUB module into the three SAGA 3D reconstructions due to their low resolutions93,204. As an alternative approach, we applied an antibody labelling method that involves incubating SAGA purified from an SGF73-TAP strain with the anti-TAP antibody. The large size and characteristic 56  shape of antibodies are more clearly distinguishable compared to GFP. We identified a large additional density corresponding to the antibody adjacent to the torso, near the proposed TAF module location (Fig 2.4D), suggesting that the DUB module shares this region with both the TAF module and parts of the SPT module. The central location of the DUB module adjacent to the conformationally flexible TAF core is consistent with its role in facilitating the donut conformation. We summarized the results of our localization studies in Fig 2.4E, and divided SAGA into regions where each module is likely located. Although EM-based labeling is a powerful technique for determining subunit locations within large complexes, it has several important limitations. The GFP tag is linked to the protein by a flexible linker, which may cause it to settle in a misleading position. Furthermore, if the tagged subunit is not globular and spans a large area, this analysis will not fully represent its location. Finally, the flexibility of SAGA complicates the comparison between tagged and untagged complexes. For these reasons, we employed cross-linking mass spectrometry analysis to examine SAGA’s molecular architecture in further detail. 2.3.7 Cross-linking mass spectrometry analysis of apo-SAGA Our EM-based localization studies enabled us to determine the spatial relationship among the different modules of SAGA. However, the relatively low resolution of these experiments precluded further understanding of the molecular organization of SAGA. Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful technique that can deduce the subunit connectivity of multi-protein complexes with precision to the amino acid residue level194. Notably, two peptides of different subunits can be cross-linked only when they are located on or adjacent to the interface between the two proteins. We applied CXMS to comprehensively map the various different subunit interfaces of the SAGA complex. We incubated SAGA purified 57  from FLAG-tagged Spt7 strain with disuccinimidyl suberate (DSS), a bifunctional cross-linker that reacts with primary amines, trypsin-digested the complex, and analyzed the resulting peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We searched the MS/MS data using the program pLink194 and identified 78 unique inter-subunit and 185 unique intra-subunit cross-links (Appendix A: Table A.1). Our cross-linking results are represented in Fig. 2.5, emphasizing the interconnectivity between modules. Very recently, Han et al applied the CXMS approach to analyze the molecular architecture of SAGA in complex with TBP121. In addition to confirming many cross-links that they identified, we were able to find interlinks involving Sgf11 and Sus1. This is likely due to better preservation of zinc finger domains in the DUB module by the FLAG affinity purification procedure in the absence of the chelating agent EGTA. Furthermore, we found unique cross-links connecting Tra1 to Spt7 and Taf6. We validated our CXMS results using available high-resolution structures of SAGA subunits, combined with the ~30 Å theoretical Cα-Cα cross-linking distance that DSS is capable of bridging204,169,70,205. We found that 20/21 cross-linked residue pairs were under 30 Å of each other, providing a high degree of confidence for the cross-links detected (Table 2.4).   Results from our CXMS analysis suggest that the TAF module in combination with the SPT subunits Spt7, Spt20, and Tra1 form a central core containing highly interconnected subunits, with the remaining subunits peripherally attached to this core. These findings are consistent with our EM-based analysis of deletion mutants and GFP localization, which suggest that the TAF and SPT modules are centrally located within the complex. Interestingly, while almost all of the SPT module subunits cross-link to members of the TAF module and Tra1, these subunits appear to constitute two distinct groups as Spt7 and Spt8 do not cross-link to Spt3 or  58   Figure 2.5. Network of SAGA subunit interconnectivity determined using cross-linking mass spectrometry. Lines denote cross-links and the line thickness is proportional to the number of cross-link spectra detected between two subunits. Cross-links involving Tra1 are shown in green for clarity. Subunits are grouped into their respective domains, as shown by the labeled highlights.  Spt20. This suggests that Spt7-Spt8 and Spt3-Spt20 are present on separate faces of SAGA, sandwiching the TAF module. On the upper edge of this ‘sandwich’, Tra1 makes contact with the two separate groups of SPT subunits, while the HAT module similarly bridges the two groups on the opposite edge. Meanwhile, aside from Sgf73, no cross-links were detected between the DUB module subunits and the rest of SAGA, suggesting that Ubp8, Sgf11, and Sus1 face outwards from the complex. Sgf73, which anchors the DUB module to SAGA, cross-links to Spt20 and Taf5, suggesting that it is situated on the Spt3-Spt20 face of the SPT-TAF-SPT sandwich.  59                    2.3.8 Interaction interface with transcriptional activators Using CXMS we generated a detailed linkage map of all SAGA’s 19 subunits. Armed with this information, we sought to probe the functional interfaces of SAGA through investigating the binding locations of transcriptional activators. We purified recombinant His-maltose binding protein (HMP)-tagged Gcn4, TATA-binding protein (TBP), and the Gal4 activation domain (Gal4AD), and showed, by pulldown experiments, that these recombinant proteins bound FLAG-purified SAGA (Fig 2.6A). We analyzed purified SAGA bound to the  Table 2.4. Validation of crosslinks detected in SAGA CXMS experiments Subunit 1 Residue Subunit 2 Residue Distance (Å)1 PDBID2 Sus1 47 Sgf11 39 13.2 3M99 Sus1 30 Sus1 43 14.2  Sus1 41 Sus1 68 11  Ubp8 2 Sgf11 39 8.8  Ubp8 441 Sgf11 63 13.5  Ubp8 15 Sgf73 33 19.8  Ubp8 22 Sgf73 40 15.1  Ubp8 22 Sgf73 33 11.5  Ubp8 22 Ubp8 116 9.9  Ubp8 315 Ubp8 389 16.7  Ubp8 326 Ubp8 363 20.2  Ubp8 326 Ubp8 395 11  Ubp8 347 Ubp8 363 21.7  Ubp8 15 Ubp8 22 11.2   Sgf29 134 Sgf29 181 8.6 3MP8 Sgf29 134 Sgf29 200 16.4  Sgf29 151 Sgf29 177 9.5  Sgf29 181 Sgf29 220 30.3*   Gcn5Br3 415 Gcn5Br 422 10.8 1E6I Gcn5Br 422 Gcn5Br 429 10.5   Gcn5HAT3 111 Gcn5HAT 153 16.6 1YGH 1Cα-Cα to distance 2X-ray structures used to measure inter-residue distance 3Gcn5 Bromodomain 4Gcn5 HAT domain 60   Figure 2.6. Localization of transcriptional activator binding sites. (A) SAGA binds the recombinantly expressed His-maltose binding protein (HMP)-tagged transcriptional activators. Amylose resin pulldowns (PD) of FLAG-purified SAGA mixed with HMP, HMP-Gcn4, HMP-TBP, HMP-Gal4 activation domain (AD) were probed using anti-FLAG antibody. FT: Flow-through, E: elution. (B)  Class averages of negative-stained SAGA bound to transcriptional activators, without glutaraldehyde cross-linking. White arrows denote additional density attributed to the HMP-tagged activators. (C) Summary of activator binding locations shown on a modular schematic of SAGA. 61   HMP-tagged activators by negative stain EM and observed that SAGA bound to Gcn4 or Gal4-AD contains additional electron densities near the globular half of SAGA’s head region near the head-torso junction (Fig 2.6B). Since Gcn4 and Gal4 are known to bind Tra1117, this observation is consistent with our proposal that Tra1 spans both halves of SAGA’s head region. We believe the slight difference in position of the extra density between Gcn4 and Gal4 can be attributed to the flexibility of the linker between the activator and the HMP tag, as opposed to binding two different sites on Tra1. Meanwhile, HMP-tagged TBP bound SAGA contains additional electron density near the shoulder region where we proposed that its binding partners, Spt3 and Spt8, are located. In contrast to the globular head region, this region undergoes a large degree of conformational rearrangement. Since the transcriptional effects of Spt3 and Spt8 binding to TBP are chromatin context-specific, the flexibility of the region may facilitate this modulation of activity. Taken together, the results from our activator binding experiments further reinforce our proposed organization of the SAGA subunits as well as revealing new possibilities in the physiological role of the complex’s flexibility. 2.3.9 Model of SAGA subunit arrangement in the context of the EM map By combining results from our EM-based GFP labeling, CXMS, and transcriptional activator binding experiments, we generated a model for the spatial arrangement of SAGA’s 19 subunits within our EM reconstruction (Fig 2.7). We approximated each subunit as spheres based on their molecular weights and the average density of proteins. The CXMS results provided distance restraints between pairs of subunits, while the localization analyses served to map specific modules to regions of density. We included two copies of Taf5, Taf6, Taf9, and Taf12 and arranged them in the same  62   Figure 2.7. Spatial arrangement of SAGA subunits. Results from EM-based subunit localization experiments and cross-linking mass spectrometry were combined with the curved SAGA 3D reconstruction to propose an overall arrangement of all 19 complex subunits. The volumes of spheres represent the molecular weight of each subunit. DUB module crystal structure PDB ID: 3M99204.   fashion as the subunits within the recently studied human TFIID core206. We were unable, however, to confirm that both copies are present in SAGA. Our TAF subunit GFP-tagging analyses did not show two additional densities within the same class average, which may be due to the alignment algorithms only focusing on one label at the expense of the other, or one label being obscured by other protein density. In spite of this, we believe that the composition of TFIID and the fact that the molecular weight of SAGA far exceeds the sum of all its subunits support the dimerization argument. The large size of the entire TAF module can be encompassed within SAGA if it is oriented along the long axis of the complex, with a small portion contained within the globular head region. This placement provides a broad interaction surface to every 63  other region of SAGA, consistent with the TAF module’s role as the backbone of the rest of the complex.  Our analysis of Tra1’s size being too large to fit within one lobe of the head led us to place spheres corresponding to its N and C terminal domains within the two regions of the head. The N-terminal domain of Tra1 consists of a long stretch of HEAT (Huntingtin, EF3, PP2A, Tor1) repeat motifs, that have the propensity to form superhelical structures thought to generate a flexible scaffold for protein binding207. Since we observed a larger degree of rearrangement in the region of the head adjacent to the shoulder, we believe that the Tra1 N-terminus localizes there. We placed the smaller, likely more globular C-terminal domain of Tra1 in the globular head region, adjacent to one arm of the TAF module. Interestingly, this region corresponds to the Gcn4 and Gal4 binding region. In an earlier ultraviolet-cross-linking study, Gcn4 was shown to be cross-linked to both Tra1 and Taf12, supporting the proposed arrangement of these two subunits within the SAGA head region208. The CXMS results suggest that within the SPT module, Spt7 resides on the opposite side of the TAF module as Spt20 and the DUB module. We positioned the DUB module, which includes the Ubp8-Sgf11-Sus1-Sgf73 N-terminal crystal structure and a sphere corresponding to the C-terminal region of Sgf73, on the Spt20 face oriented outwards, consistent with the module’s peripheral connectivity to SAGA. A recent EM structure of SAGA suggested that the DUB module localizes to a density within the torso of SAGA182. Our reconstructions do not contain a density that correspond to the one encompassing the DUB module in this other SAGA EM structure. This may be due to the gradual cross-linking of the GraFix treatment capturing a different set of conformations than the direct incubation of TAP purified SAGA with 64  glutaraldehyde used in this other study. Despite this difference, the two studies agree on the relative location of the DUB module in the context of the fully assembled SAGA complex.  Earlier EM studies on SAGA placed Spt20 to be on the end of the complex away from Tra1181, which our localization studies and other MS-based experiments argue against116,121. In our model, Spt20 is centrally located, adjacent to the SPT/TAF core, the DUB module, and Tra1. We placed Ada1 in two copies in close proximity to Taf12, as the two subunits heterodimerize through their histone fold domains209. Spt7 is thought to bind both Spt8 and the Taf10 HFD through its C-terminus210. Our placement of the Spt8 subunit within the shoulder region adjacent to Taf10 accommodates Spt7 binding to both subunits and agrees with a recent EM study of SAGA182. Furthermore, Spt3, Spt7, Spt8, and Spt20 are all known to be in close proximity to the TBP binding location121. Our own TBP binding analysis showed the binding site to be near the shoulder region along one edge of the SPT-TAF-SPT sandwich, where all of the SPT subunits have access to the activator. Both sides of the SPT module interact with Ada3, which serves as the major interface between the HAT module and SAGA. Both faces are accessible in the lower tail placement for the module, underneath the SPT-TAF-SPT sandwich. These results are in direct contention with the earlier EM-based model of SAGA, which placed the HAT module to be within the torso181. We believe that our ability to preserve the tail region in the overwhelming majority of particles contributes to a more accurate investigation of the region. Conversely, the top of the sandwich provides access between Tra1 and its many cross-linking partners. The model we generated suggests a layered arrangement of SAGA’s modules, where the top layer contains Tra1, the middle layer contains the TAF, SPT, and DUB modules, and the bottom layer contains the HAT module. 65  2.3.10 SAGA modules are transported to the nucleus individually   We then asked if the modules of SAGA are assembled individually or only in the context of SAGA. In order to address this question, we investigated if mutations that prevent SAGA modules from associating with the complex cause mislocalization of the module. Our preliminary fluorescence microscopy experiments show that ada2Δ, which dissociates the HAT module from SAGA, did not alter the nuclear localization of Gcn5-GFP (Fig 2.8A). Meanwhile, sgf73Δ, which dissociates the DUB module, causes Ubp8 to be mislocalized to the cytoplasm (Fig 2.8A). However, Sgf73 is required for the integrity of the entire DUB module121. Instead, we utilized sgf73 mutations that dissociate the module intact from SAGA (sgf73(350-400Δ)) or dissociate the module while preserving Sgf73 binding to SAGA (sgf73(2-104Δ)) (Fig 2.8B)121. Ubp8-GFP in the strain harboring the sgf73(350-400Δ) retained proper nuclear localization, while mislocalizing to the cytoplasm in the sgf73(2-104Δ)  mutant. These results indicate that the intact HAT and DUB modules are capable of its own nuclear import, and suggests that the modules assemble individually in the cytoplasm. 2.4 Discussion The large size and sophisticated composition of SAGA pose immense technical challenges to characterizing its detailed molecular structure and subunit organization in relation to its multiple roles in transcriptional regulation. Using single-particle EM, Wu et al provided the first glimpse of the overall morphology of SAGA and delineated the location of several core components of this complex181. Lee at al subsequently applied an approach combining systematic gene deletion and mass spectrometry to identify SAGA subunits that anchor the  66   Figure 2.8. Initial investigation of SAGA module localization. Strains with GFP tagged Gcn5 (HAT module) or Ubp8 (DUB module) are visualized by fluorescence microscopy to determine their cellular localizations. (A) Localization of Gcn5 and Ubp8 in ada2Δ and sgf73Δ mutants that dissociate the HAT and DUB modules, respectively. (B) Ubp8-GFP sgf73Δ strains were transformed with plasmids carrying various SGF73 mutants. sgf73(2-104Δ) dissociates the DUB module from SAGA and Sgf73, while sgf73(350-400Δ) dissociates the intact DUB module from SAGA.   67  functional modules to the core complex116. More recently, Han et al conducted CXMS analysis of SAGA in complex with TBP to determine the subunit interconnectivity121. The work presented in this paper built on and further expanded these initial efforts. In particular, through developing an improved purification method that enhanced SAGA’s stability, we were able to more precisely visualize and analyze the different conformational states of SAGA and determined the contributions of the catalytic modules in mediating structural rearrangements. Our comprehensive localization strategy, which combines EM-based labeling and CXMS of SAGA, enabled us to construct a unifying model of the subunit organization of this complex, improving our understanding of the relationships between different activities within this complex.  2.4.1 Conformational flexibility of SAGA Our improved purification procedure significantly enhanced SAGA’s stability and enabled us to systematically analyze SAGA’s conformational flexibility for the first time. In comparison to previously published EM studies of SAGA181,182, the gradual cross-linking of the GraFix method greatly preserved the presence of SAGA’s extended tail. The first SAGA EM study by Wu et al showed that the standard TAP procedure resulted in 35% of the particles not displaying the tail density181. Meanwhile, a recent study by Durand et al applied glutaraldehyde cross-linking to TAP-purified SAGA, decreasing the percentage of tail-less SAGA to 25%182. Our GraFix purified SAGA reduced the number of dissociated tails to 9%, demonstrating the effectiveness of this treatment. We proceeded to analyze the distribution of the different conformational states using single-particle EM methods. In order to investigate the degree of conformational rearrangement that SAGA undergoes, we generated 3D reconstructions of each conformation. Although our 68  reconstructions have slightly lower resolution than previously published structures181,182—likely due to the GraFix treatment making fine conformational rearrangements resolvable and therefore rendering alignment more difficult—they effectively demonstrate the extensive rearrangements between the three conformations. We suggest that the structural plasticity of SAGA reflects the need to adapt quickly to interact with different substrates and cofactors to mediate different physiological functions in the cell.   SAGA is not the only chromatin-related complex that displays a large degree of conformational flexibility. The chromatin structure remodeling complex (RSC) adopts an open and closed conformation, where a sizable domain rearranges about a cavity in a manner reminiscent of the SAGA tail211. TFIID undergoes an even greater degree of conformational rearrangement, where an entire lobe undergoes over 100 Å of movement and alters its connectivity to the rest of the complex183. Given that SAGA’s structural core is comprised largely of subunits shared with TFIID, it is tempting to speculate that the molecular mechanisms behind the conformational rearrangement might be conserved between these two related complexes. Although we were able to distinguish three major conformations adopted by SAGA, delineating the physiological roles of these conformations would require the ability to isolate SAGA locked in a distinct conformation, a technical challenge that will need to be overcome in future studies. Several factors appear to affect SAGA’s conformational flexibility, with disruption of the DUB module preventing the donut conformation from forming and the removal of the HAT module causing the shoulder region to be much more mobile. Disruption of the DUB module decreases HAT activity while only marginally affecting its structural integrity. Interestingly, it has been shown that the acetyltransferase and deubiquitination activities of SAGA show 69  significant crosstalk, interacting both genetically and catalytically56,212. Our finding that the absence of the DUB module affects the flexibility and presence of SAGA’s tail region, where the HAT module is located, may reveal an indirect co-operativity between the two catalytic activities. 2.4.2 Spatial arrangement of SAGA chromatin-binding domains We combined our 3D SAGA reconstructions with subunit localization and interconnectivity data to generate a model detailing the spatial organization of all 19 SAGA subunits. We show that SAGA is likely arranged into three major layers, with the topmost layer housing Tra1, the middle layer containing the SPT-TAF-SPT sandwich and the DUB module, and the lower layer encompassing the HAT module. This layered arrangement of SAGA is supported by CXMS data from Han et al, and our analyses, with very few subunits bridging the top and bottom layers. Although our modeling approach relies heavily on CXMS data for pairwise subunit arrangements, we recognize that the technique has a number of limitations. Although each detected crosslink directly corresponds to two lysine residues adjacent to each other, the lack of crosslinks does not necessarily mean that two subunits are not positioned close to each other. Lysine pairs that are inaccessible to the solvent will not generate crosslinked peptides. This is particularly relevant in extensive interaction interfaces, where buried lysines will likely not be crosslinked. Even with these considerations, however, we showed that detected crosslinks are a reliable indicator of proximity that is extremely useful for our modeling approach. Based on the model we constructed, subunits with chromatin-binding domains are clustered along one side of the complex in close proximity to each other (Fig 2.9). These domains, including the Gcn5 and Spt7 bromodomains, Sgf29 Tudor domain, Ada2 SANT and 70  SWIRM domain, and the Spt8 WD40 domain, all of which have been shown to bind different chromatin templates23. Their proximity suggests a major interaction surface with the chromatin template within a region of SAGA that shows a large degree of flexibility. Chromatin surrounding a transcribed gene is by definition dynamic, decorated with various post-translational modifications depending upon the state of the cell. SAGA’s activity transitions between the different phases of transcription, from acetylation during transcription initiation to deubiquitination during elongation. SAGA’s highly diverse chromatin binding domains are capable of binding methylated and acetylated histones, suggesting considerable versatility in template recognition. Given that transcription involves extensive remodeling of nucleosome occupancy, SAGA must be able to compensate for variations in nucleosome positions. The extreme diversity of context-specific activities that SAGA fulfills, compounded with the recent proposition that the complex is active in all RNA polymerase II-mediated transcription213, provides a compelling hypothesis for a highly flexible and adaptable chromatin  Figure 2.9. Chromatin-binding domains of SAGA cluster around one edge of the complex. SAGA subunits with domains known to bind histones are shown in the spatial arrangement model.   71  interaction surface. In contrast, our finding that the activator binding surface is relatively static is likely due to the conserved acidic patches within the activation domains of transcriptional activators serve as universal adapters, delegating the task of chromatin binding to the activators’ DNA binding domains117. These hypotheses on the nature of SAGA flexibility predict that other chromatin-binding complexes will often be flexible; a feature that is often de-emphasized in single-particle EM studies where this property negatively affects achievable resolution. We believe that conformational variability of chromatin-binding complexes should be studied more closely, as such studies could provide intriguing new insights into the mechanism of action of these important regulators.  SAGA is a fascinating multifunctional complex that provides a paradigm to delineate the molecular mechanisms of fundamental transcriptional processes and to gain insights into how important chromatin-modifying complexes that exert exquisite epigenetic control over all eukaryotic gene expression. Further understanding of SAGA’s mechanisms of action would require higher resolution analysis of the full complex and individual subunits, using a joint approach of various structural techniques. 2.4.3 Site of SAGA assembly In this study we began to address the question of how SAGA is assembled in the cell. Since it is highly unlikely that all 19 SAGA subunits carry nuclear localization signals, we hypothesized that proper nuclear transport requires some degree of assembly within the cytoplasm. If SAGA fully assembles in the cytoplasm, then we expect mutations that decouple subunits from the complex to result in defective nuclear localization. Our initial experiments focused on the HAT and DUB modules of SAGA, two modules that can be dissociated from the complex through knockouts of ADA2 and SGF73, respectively. We show that as long as both 72  modules are intact, they do not require association with SAGA for nuclear localization. These results suggest that these modules assemble in the cytoplasm, and the intact SAGA within the nucleus. Since subunits from both the HAT and DUB modules can act independently of SAGA75,125, their independent recruitment to the nucleus is unsurprising. Whether the relative abundance of different HAT complex modules within the nucleus result in a different HAT complex demographic is an intriguing question that remains unanswered.     73  Chapter 3: Modular organization of the essential yeast lysine acetyltransferase NuA4 3.1 Introduction Esa1 is the only essential KAT in yeast and acts as the catalytic subunit of the nucleosomal acetyltransferase of histone H4 (NuA4) complex214–216. NuA4 acetylation has been implicated in multiple cellular processes such as transcription of ribosomal genes and DNA double-strand break repair137,217,218. In addition to histone acetylation, NuA4 also acetylates non-histone proteins with key roles in metabolism, transcription, cell cycle progression, RNA processing, and autophagy142. Its human homologue, the Tip60 complex, has been identified as a tumor suppressor219. Despite its importance, relatively little is known about the structure of the NuA4. NuA4 is a 1.0 MDa complex consisting of 13 subunits that organizes into four functional modules: the Piccolo NuA4, Eaf5/7/3, shared SWR1C, and the recruitment modules (Table 3.1)143,216,133. Piccolo is responsible for NuA4 KAT activity, Eaf5/7/3 facilitates RNA Pol II binding and assists transcription elongation, the recruitment module mediates transcription activator targeting of NuA4, and the shared SWR1C module consists of subunits shared with the SWR1 complex45,133,135,136. We sought to understand how these modules function cooperatively by investigating their spatial organization within the complex. A study published by Chittuluru et al presented a structure of NuA4 obtained by electron microscopy. It identified the overall structure of NuA4 and determined the locations of the Epl1 subunit and nucleosome binding site200. We sought to complement this study by localizing the other NuA4 modules by single particle EM and determining the intersubunit connectivity within the complex.   74  Table 3.1. Subunits of yeast NuA4 Module Subunit MW (kDa) Piccolo NuA4 Eaf6 12.9   Yng2 32.1   Esa11 52.6   Epl12 96.7 Eaf5/7/3 Eaf52 31.6   Eaf3 45.2   Eaf7 49.4 Recruitment Tra1 433.2 SWR1C Yaf9 26.0   Act1 41.7   Arp4 54.8   Swc4 55.2   Eaf1 112.5 1Catalytic subunit    2Anchors module to complex      3.2 Experimental methods 3.2.1 S. cerevisiae strain construction Standard S. cerevisiae genetics and culturing methods were used (See 2.2.1). A list of strains and plasmids used in this study is provided in Table 3.2. 3.2.2 Purification of NuA4  For each NuA4 purification, 4-8L of S. cerevisiae strains expressing a C-terminally FLAG-tagged Epl1 were grown to an OD600 of ~4 and harvested by centrifugation. Cell pellets were lysed using the SPEX 6870 freezer mill (SPEX SamplePrep LLC, Metuchen, NJ) under      75   liquid nitrogen temperatures. Cell pellets were resuspended in lysis buffer in a 1:2 ratio. Initial purifications of NuA4 utilized an identical lysis buffer to those used for SAGA (40 mM HEPES pH 7.4, 350 mM NaCl, 0.1% Tween-20, 10% glycerol, 1 mM PMSF, 50 mM NaF, 0.1 mM Na3VO4, 2 mM benzamidine, and cOmplete EDTA-free protease inhibitor [Roche]). Meanwhile, optimized purifications utilized an alternate buffer: 40 mM HEPES pH 7.4, 350 mM NaCl, 0.1% CHAPS, 10% glycerol, 1 mM magnesium acetate, 1 mM PMSF, 50 mM NaF, 0.1 mM Na3VO4, 2 mM benzamidine, and cOmplete EDTA-free protease inhibitor. Lysates were precleared by ultracentrifugation at 154,000 xg for 30 minutes. The cleared lysate was incubated with 500 μL anti-FLAG M2 resin (Sigma-Aldrich, St. Louis, MO) at 4ºC for 1 hour. The resin was washed three times with wash buffer (40 mM HEPES pH 7.4, 350 mM NaCl, 0.1% CHAPS, 10% glycerol, 1 mM magnesium acetate), then incubated with 2.5 μg/mL RNAse A for 30 minutes at 4ºC. The resin was washed another three times with wash buffer, and bound proteins were eluted by incubation with 2x 500 μL elution buffer (Wash buffer containing 500 μg/ml 3x FLAG peptide [GenScript, Piscataway, NJ]) for 15 minutes. Eluted proteins were then subjected to gradient fixation (GraFix)188: Eluates were loaded onto 15-30% glycerol gradients with or Table 3.2. Strains used in this study of NuA4 STRAINS     Name of Strain Genotype Origin Integration plasmid Plasmid origin BJ1991 MAT-α pep4-3 leu2 trp1 ura3-52 prb1-1122 gal2 J. Rubinstein - - BJ1991 Epl1-FLAG BJ1991 EPL1-FLAG::KanMX6 This study p3FLAG-KanMX6 M. Kobor BJ1991 Epl1-FLAG Arp4-GFP BJ1991 EPL1-FLAG::KanMX6 ARP4-GFP::URA3 This study pFA6a-eGFP-URA3 (pKT0209) K. Thorn220 (Addgene) BJ1991 Epl1-FLAG Yaf9-GFP BJ1991 EPL1-FLAG::KanMX6 YAF9-GFP::URA3 This study pFA6a-eGFP-URA3 (pKT0209) K. Thorn (Addgene) BJ1991 Epl1-FLAG Esa1-GFP BJ1991 EPL1-FLAG::KanMX6 ESA1-GFP::URA3 This study pFA6a-eGFP-URA3 (pKT0209) K. Thorn (Addgene) 76  without 0-0.05% glutaraldehyde and ultracentrifuged at 76,000x g for 16 hours. Gradients were fractionated using the Gradient Station (BioComp, Fredericton, Canada). Fractions from gradients without crosslinker were TCA (TriChloro Acetic acid) precipitated and analyzed by SDS-PAGE. 3.2.3 NuA4 buffer optimization  Approximately 100 μL volume of ground yeast containing FLAG-tagged Epl1 was split to 16 1.5 mL tubes. Cells in each tube was resuspended in 200 μL different buffer variants (Fig 3.2) of the standard purification buffer (350 mM NaCl, 40 mM HEPES-NaOH pH 7.4, 10% glycerol, and 0.1% Tween-20). Buffers containing different buffering agents, salts, or detergents substitutes the in the new component. Lysates were centrifuged in a microcentrifuge for 5 minutes at 4,000 rpm, and supernatants were again centrifuged for 30 minutes at 14,000 rpm. 2 μL of supernatant and resuspended pellet (50 μL of corresponding buffer) were dotted onto nitrocellulose (Bio-Rad). The nitrocellulose strip were then analyzed by Western Blot using an α-FLAG (Sigma) and α-mouse-horseradish peroxidase antibodies (Sigma). Signal was visualized using enhanced chemiluminescence reagent (GE) and X-ray film (Mandel). Small scale pulldowns were done identical to above (3.3.2), but using 2.7 g of freeze-ground Epl1-FLAG yeast. 3.2.4 Electron microscopy Fractions containing ~10 μg/ml NuA4 were applied to glow discharged carbon coated grids (Ted Pella, Redding, CA) and stained with uranyl formate as described previously171. Grids were imaged using a Tecnai Spirit G2 (FEI, Hillsboro, OR) at an accelerating voltage of 120 kV. Micrographs were acquired at a nominal magnification of 49,000x with an FEI Eagle 4k charge-coupled device camera using a defocus of 1-1.5 μm.  77  3.2.5 Image processing For the 3D reconstruction of NuA4, 2 x 2 micrograph pixels were averaged for a final pixel size of 4.7 Å/pixel and phase-flipped CTF corrected using CTFFIND3 and SPIDER190,221. 200 particles were picked manually, then classified, aligned, and used to generate class averages in RELION222. These class averages are used as templates for autopicking for a final particle count of 16,776. These particles were subjected to 2D classification, and several class averages were used to generate an initial model in EMAN2223. NuA4 particles were used for 3D classification using RELION. All the classes have similar overall morphology, and the entire dataset was used for 3D refinement to yield a final resolution of 35 Å using the 0.143 FSC criterion. Details on this process can be found in Figure 3.3. 3.2.6 Chemical crosslinking coupled to mass spectrometry FLAG eluates of NuA4 were incubated with either 40, 80, 160, or 240 μM of disuccinimidyl suberate (DSS) for 30 minutes are room temperature. The crosslinking reaction was quenched by adding 50 mM Tris-HCl pH 8.0 for 15 minutes. Cross-linked samples were analyzed on 5-20% SDS-PAGE gels (BioRad, Hercules, CA) followed by Coomassie blue G-250 staining. High molecular weight bands appearing in the crosslinked samples were excised and processed for mass spectrometry analysis as previously described224.  3.2.7 Modeling NuA4 architecture  The three-dimensional reconstruction of NuA4, X-ray structures of Act1, Eaf3, Arp4, Yaf9, and Esa1 (PDB IDs: 1YAG225, 3E9G226, 3QB0227, 3RLS, and 3TO687), and homology models of Tra1C, Swc4 SANT, Eaf3, Eaf5C, Yng2 were used to generate a model of complex architecture using UCSF Chimera191. Subunits were placed manually based on EM-labeling and CXMS experiments. Spheres of radii proportional to molecular weight were used to approximate 78  subunits without high resolution structure information. The cryoEM reconstruction of Piccolo (EMD-953689) was placed based on the approximated position of the module. The orientation was deliberately positioned to propose the possibility for simultaneous contact of the nucleosome with lobes 2 and 3 of NuA4. 3.2.8 Analysis of human TIP60 complex  Dr. Jacques Côté provided FLAG eluates of human Tip60 complexes prepared as described previously228. The eluates were subjected to GraFix using identical spin conditions to NuA4. Microscopy and image processing conditions were identical to the NuA4 reconstruction. The final reconstruction utilized 12,019 particles for a final resolution of 39 Å using the gold-standard FSC criterion at a cutoff of 0.143. 3.3 Results and discussion 3.3.1 Purification of intact NuA4 from S. cerevisiae We purified NuA4 from an S. cerevisiae strain expressing C-terminally FLAG-tagged Epl1 using FLAG affinity purification coupled with gradient fixation (GraFix188) in identical conditions as SAGA and visualized it by single particle negative stain electron microscopy (Fig 3.1). In order to optimize our crosslinking protocol, each glutaraldehyde-containing gradient is paired with an identical gradient without crosslinker. Class averages of particles from the gradient without crosslinker showed, as expected, a complex with a large degree of similarity to the head of SAGA (Fig 3.1C,D). However, the class averages also contained a prominent additional density not observed in the previous EM study extending out from one end of the complex (Fig 3.1D, white triangle)200. Surprisingly, however, class averages from the crosslinked complex were more heterogeneous and at times contained additional densities (Fig 3.1E). In many cases, these additional densities are blurred, which is characteristic of either 79  conformational or compositional heterogeneity. In order to improve our single particle analysis, we sought to optimize the NuA4 purification conditions. We focused on improving the purification buffers for the complex. In order to test multiple buffer conditions, we partitioned equal amounts of freeze-ground Epl1-FLAG yeast and resuspended each in a series of buffers with one variable component. We then cleared the lysate by high speed ultracentrifugation and analyzed both the pellet and supernatant by dot blotting using an α-FLAG antibody (Fig 3.2A). All of the pellet dots and most supernatant dots contained too little Epl1-FLAG for a clear signal. However, we observed clear supernatant signal from buffers containing CHAPS detergent and magnesium acetate (MgOAc) (Fig 3.2A). Small scale FLAG affinity purifications also show improved purification yields, particularly for the low molecular weight subunits (Fig 3.2B). We then utilized buffer containing both CHAPS and MgOAc in a full scale NuA4 purification (Fig 3.2C). Single particle analysis of NuA4 purified from the modified buffer revealed class averages with substantially reduced heterogeneity, particularly in two prominent lobes that were not characterized in the previously determined NuA4 structure (Fig 3.2D). These additional lobes may correspond to labile modules that dissociate upon protein purification or EM grid preparation in the unoptimized buffer.  3.3.2 Three dimensional reconstruction of NuA4 and subunit localization We proceeded to generate a three dimensional (3D) reconstruction of intact NuA4 (Fig 3.3, 3.4). The three lobes observed in the 2D analysis of NuA4 are clearly represented in the reconstruction. The lobes are arranged in a plane and project outwards from the centre of the complex. Lobe 1 clearly resembles the ‘head’ region of SAGA and forms a hollow cradle-like shape characteristic of PIKK proteins202,229. Lobes 2 and 3 form globular densities that are bridged to the rest of NuA4 through relatively slender connections, which may correspond to 80    Figure 3.1. Purification and visualization of yeast NuA4. NuA4 was purified from strains containing 3xFLAG-tagged Epl1 using affinity purification and glycerol gradient ultracentrifugation. (A) Silver-stained SDS-PAGE of fractions from a glycerol gradient without crosslinker. Lane marked with a * is used for negative stain electron microscopy (EM) analysis. (B) Representative micrograph of purified NuA4. Scale bar corresponds to 100 nm. (C) The previously published negative stain class average of NuA4 by Chittuluru et al (2008) beside a class average of SAGA. (D) Representative class averages of NuA4 purified without crosslinking. White triangle denoting the previously unobserved density. (E) Representative class averages of NuA4 purified with glutaraldehyde crosslinker included in the glycerol gradient (GraFix). White triangles highlights another additional density not observed without crosslinking. Scale bars in C and D correspond to 100 Å.  81   Figure 3.2. Optimizing NuA4 purification buffers. (A) Dot blot optimization of different buffer conditions. Equal amounts of freeze ground pellets of Epl1-FLAG yeast were resuspended in a series of buffers and centrifuged. Both supernatant (Sup) and pellet (Pel) fractions were analyzed by dot blotting with an α-FLAG antibody. Buffer compositions are indicated in the lower panel. Buffers indicated by an S is identical to the standard buffer. (B) Affinity pulldowns of NuA4 using the modified buffers. (C) SDS-PAGE of glycerol gradient fractions from a full scale purification of NuA4 using buffer containing both 0.1% CHAPS detergent and magnesium acetate (MgOAc). Further EM analysis were conducted on GraFix-stabilized NuA4 from the fraction corresponding to 82  the * fraction. Band corresponding to Eaf5‡ is not visible. (D) Representative 2D class averages of GraFix-stabilized NuA4 purified using the modified buffer. Scale bar corresponds to 100 Å. different modules within the complex.  In order to identify the different densities observed in the NuA4 reconstruction, we pursued an EM-based subunit localization strategy. We purified NuA4 from various yeast strains    Figure 3.3. Mapping of NuA4 lobes and subunits. (A) 3D reconstruction of the curved conformation of SAGA. (B) 3D reconstruction of intact NuA4, with specific lobes labeled in the ‘top’ view. Scale bar corresponds to 100 Å. (C) Class averages of NuA4 complexes purified from yeast strains containing mutated EAF5 (eaf5Δ), which dissociates the Eaf5/7/3 module, C-terminally truncated EPL1 [epl1(1-495)], which dissociates the Piccolo module, or GFP-tagged Arp4, Yaf9, or Esa1. White triangles indicate additional GFP densities.  83    Figure 3.4. 3D reconstruction of NuA4. (A) Initial model of NuA4 generated de novo in EMAN2 from 2D class averages of NuA4. (B) 3D classification of NuA4 particles conducted in RELION. Due to the similarities of the overall shapes of the classes, no particles were discarded. (C) Class 4 and all NuA4 particles were used in the 3D auto-refinement step. (D) Gold-standard Fourier shell correlation curve showing a reconstruction resolution of 35 Å. (E) Euler plot showing the orientational coverage of the particles used in generating the reconstruction. containing either GFP-tagged subunits or subunit deletions and compared their 2D class averages with wild-type NuA4. NuA4 isolated from eaf5Δ mutants, which has been shown to dissociate the entire Eaf5/7/3 module from the complex135, resulted in disruption of lobe 3. Complexes containing GFP-tagged Arp4 and Yaf9, both components of the module shared with the SWR1 84  complex (shared SWR1C module), displayed additional density centred on lobe 2 when  compared to wild-type NuA4 (Fig 3.3C). We attempted to localize the Piccolo module of NuA4 through GFP-tagged Esa1 and found additional density adjacent to the central region of the complex (Fig 3.3C). When we introduced a C-terminal truncation to Epl1, which dissociates Piccolo from NuA4, and purified the complex using Eaf1-FLAG, we observed a large disruption to the integrity of the complex (Fig 3.3C). The extensive effects of Piccolo module mutations to the stability of NuA4 suggests a central location of the module.  3.3.3 Crosslinking coupled to mass spectrometry analysis The heterogeneity and limited availability of purified NuA4 precluded high resolution structure determination using techniques such as cryoelectron microscopy. Instead, we utilized chemical crosslinking coupled to mass spectrometry (CXMS) to identify the subunit interaction map and complement our negative stain EM analysis. We incubated purified complexes with the bifunctional lysine crosslinker disuccinimidyl suberate (DSS) and identified crosslinked subunits through liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified 97 and 143 total intersubunit and intrasubunit crosslinks, respectively (Fig 3.5, Appendix A: Table A.2). Validation of 10 intrasubunit crosslinks using available high resolution structures showed complete agreement, with all lysine pairs analyzed being within the ~30 Å theorized maximum crosslinking distance205. Overall, subunits within each NuA4 module are extensively crosslinked with each other while intermodular connectivity is centred on an anchoring subunit associating with Tra1, Eaf1, or Epl1.  Many of the observed crosslinking patterns are consistent with previous findings. Eaf1, the only subunit unique to NuA4, serves as a key scaffold for the complex and contains a high 85  number of intersubunit crosslinks. It is positioned very closely to Tra1, showing extensive crosslinking with the FAT (FRAP-ATM-TRRAP) and FRB (FKBP12-Rapamycin-Binding) domains. Mutational analysis of this region of Tra1 suggested that it is important for the integrity of both SAGA and NuA4130. Most of the Eaf1-Tra1 crosslinks are centred about the C-terminus of Eaf1. The Eaf1 SANT domain, which is essential for this interaction, is also found within this extensively crosslinked region143. Eaf1 and Tra1 also associate with Epl1 and Eaf5, which serve as anchors for their respective modules134.  Figure 3.5. Crosslinking coupled to mass spectrometry of NuA4. (A) Purified NuA4 was incubated with disuccinimidyl suberate and analyzed by mass spectrometry. Crosslinked residues between different subunits are shown by black lines. Tick marks indicate 100 residues. (B-D) Crosslinks between the subunits of the Piccolo, shared SWR1C, and Eaf5/7/3 modules, respectively.  86   Within Piccolo, we observe extensive crosslinking of the Epl1 C-terminus to the rest of the primary scaffolding subunits of NuA4 (Tra1-Eaf1-Epl1). Conversely, other Piccolo subunits crosslinked to the N-terminus of Epl1. These observations are consistent with the finding that C-terminal truncation of Epl1 releases intact Piccolo from NuA4134. The shared SWR1 complex module seems to be primarily anchored through Swc4, whose N- and C-termini crosslinking near the Eaf1 HSA domain and the central region of Epl1, respectively.  3.3.4 Model of NuA4 subunit organization We combined electron microscopy, crosslinking mass spectrometry, and biochemical data to propose a model for NuA4 subunit architecture (Fig 3.6A). Lobe 1 serves as the main scaffold of the complex and encompasses Tra1, the Piccolo module, and Eaf1. Lobe 2 consists of the SWR1C shared module and is anchored to lobe 1 primarily through Swc4, though the two  Table 3.3. Validation of crosslinks detected in NuA4 CXMS experiments Subunit Residue 1 Residue 2 Distance1 PDBID2 Esa1 330 432 7.4 3TO6 Arp4 210 219 4.2 3QB0   218 227 16.1     259 296 19.8     313 323 26     323 326 9   Eaf3 108 116 12.5 3E9G   109 116 10.4     109 119 15.3     116 120 6.2   1Cα-Cα distance 2X-ray structures used to measure inter-residue distance 87   Figure 3.6. Model of NuA4 subunit organization. (A) Model of NuA4 subunit organization. High resolution structures (PDB IDs: 1YAG225, 3E9G226, 3QB0227, 3RLS, and 3TO687) or homology models (Tra1C, Swc4 SANT, Eaf3, Eaf5C, Yng2) of NuA4 subunits were arranged based on electron microscopy and crosslinking coupled to mass spectrometry information. Subunits without structure information were substituted with spheres proportional to their sizes. (B) The 7.9 Å resolution cryo-electron microscopy structure of Piccolo (EMD-953689) bound to a nucleosome mapped onto the proposed NuA4 model. Subunits containing chromatin reader domains in the vicinity of the nucleosome are indicated. lobes contact each other at multiple sites. Lobe 3, which consists of the Eaf5/7/3 module, is much more peripherally associated with lobe 1 anchored entirely through Eaf5. A recent study determined the crystal structure of a portion of the Piccolo NuA4 module, as well as a 7.9 Å 88  cryo-EM structure of the module bound to a nucleosome89. We approximated the nucleosome-bound structure onto our model to determine its orientation relative to NuA4 (Fig 3.6B). Strikingly, the nucleosome snugly fits within the space cradled by the Piccolo, shared SWR1C, and Eaf5/7/3 modules. In this arrangement, subunits with chromatin reader domains (Esa1, Yng2, Swc4, Eaf3, Eaf1, Yaf9) are all in close proximity to the nucleosome (Fig 3.6B). Although the position and orientation of these subunits are likely imprecise, this configuration suggests that all of NuA4’s chromatin-interacting domains can interact with the nucleosome simultaneously. Conversely, these domains may redundantly position NuA4 over different nucleosomal templates, only engaging a subset of chromatin readers at any given time. This study provides the first glimpse into the subunit organization of the intact NuA4 complex. In order to cement our understanding of NuA4’s structure, we need to determine a higher resolution reconstruction through techniques like cryo-EM. Such a high resolution structure, especially with bound nucleosomes, will elucidate how chromatin-modifying complexes containing multiple chromatin-interacting domains engage their substrates. 3.2.5 Initial characterization of the human TIP60 complex NuA4 is highly conserved from yeast to mammals, and its homologue in humans is the TIP60 complex133. One peculiar aspect of the Tip60 complex is that it seems to be a fusion of both the yeast NuA4 and SWR1 complexes. Since NuA4 KAT activity stimulates H2A.Z deposition by SWR1230, the fusion of these two complexes may streamline the action of these two catalytic activities. We collaborated with Dr. Jacques Côté, whose group successfully purified human TIP60228, investigating the structure of the intact complex. Initial EM analysis revealed a large complex roughly the size of SWR1 and NuA4 combined (Fig 3.7). Strikingly, class averages of TIP60 resemble the class averages of NuA4 and SWR1 (Watanabe et al 2015) 89  stacked on top of each other (Fig 3.7)231. The position of lobe 2, which is shared between the two complexes, at the junction of the two halves of TIP60 is reminiscent of a ‘trailer hitch’ that binds the complex together. Initial 3D reconstruction also recapitulate the resemblance, though additional data collection and model refinement is necessary for higher quality models. High resolution studies of TIP60 will further elucidate its mechanism of action.      Figure 3.7 Initial EM analysis of the human Tip60 complex. (A) Class averages of NuA4 and SWR1C (From Watanable et al). (B) Class averages of the human Tip60 complex C. 3D reconstruction of the human Tip60 complex Scale bars in this figure corresponds to 100 Å.  90   Figure 3.8. 3D reconstruction of TIP60. (A) Initial model of TIP60 generated de novo in EMAN2 from 2D class averages. (B) 3D classification of TIP60 particles conducted in RELION. (C) The model from class 2 and the particles from classes 1, 2, and 4 were used in the 3D auto-refinement step. (D) Gold-standard Fourier shell correlation curve showing a reconstruction resolution of 38 Å. (E) Euler plot showing the orientational coverage of the particles used in generating the reconstruction.  91  Chapter 4: Molecular architecture of the yeast Elongator complex 4.1 Introduction The Elongator complex is an evolutionarily conserved multi-subunit protein complex initially identified as a component of the elongating form of RNA polymerase II232. Subsequent studies revealed that Elongator participates in various processes, including histone acetylation76, transcription regulation233,234, α-tubulin acetylation147, and tRNA modification80. In particular, Elongator plays an essential role in the addition of 5-methoxycarbonylmethyl and 5-carbamoylmethyl to the wobble base pair of transfer RNAs (tRNAs) that stabilizes codon-anticodon interactions and facilitates translation79. Because of its broad range of activities, deficiencies in human Elongator have been shown to give rise to serious pathological conditions such as familial dysautonomia and other neurological disorders235–238.  Elongator consists of six unique subunits (Elp1, Elp2, Elp3, Elp4, Elp5, Elp6), with two copies of each protein associating into a dodecameric ~850 kDa holo-complex148,155. Previous biochemical studies have shown that the Elongator subunits are organized into two subassemblies: one composed of Elp1, Elp2, Elp3 (Elp123), and the other one composed of Elp4, Elp5, Elp6 (Elp456)148,239. Elp123 houses Elp3, the main catalytic subunit, which contains both a Gcn5-related N-acetyltransferase histone acetyltransferase (HAT) domain and a radical S-adenosylmethionine (SAM) domain114. Elp456 forms a heterohexameric RecA-like ATPase ring, which possesses ATP-modulated tRNA binding activity155. How the multiple catalytic domains of Elongator are regulated is poorly understood.  Despite a fairly extensive understanding of the domain composition of Elongator and recent advances in delineating the structural properties of individual Elongator subunits (eg. Elp1 C-terminal domain, Elp2, and Elp456)111,154–156, the overall architecture and subunit organization 92  of this complex remain unclear. We have isolated the holo-Elongator complex from yeast Saccharomyces cerevisiae and obtained the first structural data on this complex using an integrative approach combining single particle electron microscopy (EM), crosslinking coupled to mass spectrometry (CXMS), and multi-scale modeling. Our data revealed an unexpected asymmetric overall architecture and subunit organization of Elongator, and provided a framework to understand the molecular basis of Elongator’s multifunctionality.   4.2 Experimental methods 4.2.1 Yeast methods and strain construction Standard S. cerevisiae genetics and culturing methods were used (See 2.2.1). A list of strains and plasmids used in this study is provided in Table 4.4. 4.2.2 Purification of yeast Elongator To isolate the yeast holo-Elongator complex, 4 L of S. cerevisiae strains expressing C-terminally FLAG-tagged Elp1 were grown to an OD600 of ~4, harvested, and lysed by freeze grinding using a SPEX 6870 freezer mill (SPEX SamplePrep LLC, Metuchen, NJ). Freeze-ground yeast were resuspended in lysis buffer (40 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 50 mM NaF, 0.1 mM Na3VO4, 2 mM benzamidine, and cOmplete EDTA-free protease inhibitor [Roche]) and pre-cleared by ultracentrifugation at 154,000 xg for 30 minutes. Clarified lysate was incubated with 500 μL anti-FLAG M2 resin (Sigma-Aldrich, St. Louis, MO) at 4°C for 1 hour. The resin was collected and washed 3 times with lysis buffer without inhibitors, incubated with 2.5 μg/ml RNase A for 30 minutes at 4°C, and washed 3x with lysis buffer without inhibitors. Bound Elongator was eluted with 2x 500 μL of elution buffer (lysis buffer without inhibitors containing 500 μg/mL 3x FLAG peptide (GenScript, Piscataway, NJ). Further purity was achieved by subjecting the FLAG eluate to  93    Table 4.1. List of strains and plasmids used in this study of Elongator STRAINS     Name of Strain Genotype Origin Integration plasmid Plasmid origin BJ1991 MAT-α pep4-3 leu2 trp1 ura3-52 prb1-1122 gal2 J. Rubinstein - - BJ1991 Elp1-FLAG BJ1991 ELP1-FLAG::KanMX6 This study p3FLAG-KanMX6 M. Kobor BJ1991 Elp1-FLAG Elp2-MBP BJ1991 ELP1-FLAG::KanMX6 ELP2-MBP::URA3 This study pFA6a-MBP-URA3 This study BJ1991 Elp1-FLAG Elp3-MBP BJ1991 ELP1-FLAG::KanMX6 ELP3-MBP::URA3 This study pFA6a-MBP-URA3 This study BY4741 MAT-a his3-1 leu2-0 met15-0 ura3-0 ATCC - - BY4741 Elp4-FLAG BY4741 ELP4-FLAG::KanMX6 This study p3FLAG-KanMX6 M. Kobor BY4741 Elp4-FLAG elp1Δ BY4741 ELP4-FLAG::KanMX6 elp1Δ::HIS3MX6 This study pFA6a-HIS3MX6 V. Measday BY4741 Elp4-FLAG elp2Δ BY4741 ELP4-FLAG::KanMX6 elp2Δ::HIS3MX6 This study pFA6a-HIS3MX6 V. Measday BY4741 Elp4-FLAG elp3Δ BY4741 ELP4-FLAG::KanMX6 elp3Δ::HIS3MX6 This study pFA6a-HIS3MX6 V. Measday NCYC 1368 Kluyveromyces lactis - NCYC - - T7 Express E. coli - NEB - This study PLASMIDS     Name of Plasmid ORF Origin Other name  pRS416 - L.J. Howe -  pRS416-ELP2-3xHA ELP2-3xHA* This study ELP2-3xHA  pRS416-ELP2(EKI459-461AAA)-3xHA ELP2(EKI459-461AAA)-3xHA This study elp2-1  pRS416-ELP2(LTIT607-610AAAA)-3xHA ELP2(LTIT607-610AAAA)-3xHA This study elp2-2  pRS416-ELP2(RDR626-628AAA)-3xHA ELP2(RDR626-628AAA)-3xHA This study elp2-3  pRS416-ELP2(RIIW654-657AAAA)-3xHA ELP2(RIIW654-657AAAA)-3xHA This study elp2-4  pQLink-ELP4-ELP5-6xHis-ELP6 ELP4, ELP5, 6xHA-ELP6 This study -  * ELP2 ORF includes 500 bp upstream and 100 bp downstream sequences from the genome 94  glycerol gradient ultracentrifugation. More specifically, 200 μL of eluate was overlaid onto a linear 15-30% glycerol gradient, ultracentrifuged at 76,000x g using an SW 55 Ti rotor (Beckman Coulter, Brea, CA), and fractionated using the Gradient Station (BioComp, Fredericton, Canada). For the final 3D reconstruction of Elongator, we utilized the GraFix method to enhance stability of the purified complex, with a glutaraldehyde gradient of 0-0.05% added to the glycerol gradient188. The initial random conical tilt model was prepared using Elongator particles isolated by glycerol gradient ultracentrifugation without glutaraldehyde. Preparation of Elongator in low salt conditions utilizes an identical purification procedure, except all buffers contains 75 mM NaCl as opposed to 150 mM. Preparation of the Elp123 subcomplex utilizes an identical FLAG pulldown procedure to that of the intact complex using Elp1-FLAG yeast, except all buffers used contain 300 mM NaCl. The FLAG eluate was concentrated using a 100 kDa cutoff centrifugal filter unit (Merck Millipore, Billerica, MA) and further purified by size exclusion chromatography using a Superose 6, 10/300 column (GE Healthcare, Little Chalfont, UK). The purification of Elongator with subunit deletions utilized the same general procedure, except that for these experiments only 1 L of yeast was used and lysis was performed using a coffee grinder (Krups, Solingen, Germany). Furthermore, the FLAG eluates were directly used for mass spectrometry analysis. 4.2.3 Antibody labeling analysis GraFix-purified Elongator containing an Elp1 C-terminal FLAG tag was incubated with 50 μg/mL α-FLAG M2 antibody (Sigma-Aldrich, St. Louis, MO) for 10 minutes at room temperature.  95  4.2.4 Electron microscopy The peak glycerol gradient fractions containing ~10 μg/ml Elongator were adsorbed to glow discharged carbon coated grids and stained uranyl formate as described previously171. The EM specimens were examined using a Tecnai Spirit G2 (FEI, Hillsboro, OR) operated at an accelerating voltage of 120 kV. Micrographs were acquired at a nominal magnification of 49,000x with an FEI Eagle 4K charge-coupled device (CCD) camera at a defocus of 1-1.5 μm. For collecting tilt pair data for determining an initial 3D model, the same area of the grid was imaged at 60° tilted and untilted.  4.2.5 Image processing To determine the 3D reconstruction of Elongator, we first obtained an initial model using the random conical tilt approach implemented in the SPIDER software suite190. In brief, tilt pair particles from the tilt pair dataset were selected using WEB, with the untilted particles subjected to alignment and classification. Tilted particles from the most populated classes were used to calculate a reconstruction using the back projection procedure.  For the final Elongator reconstruction, we collected 100 micrographs which were phase-flip CTF (contrast transfer function) corrected using CTFFIND3 and SPIDER (final pixel size 7.0 Å/pixel)190,221.  The micrographs were subjected to template-based autopicking in RELION to obtain a total of 17,074 particles222. These particles were then aligned and classified using maximum likelihood methods and the averages for each class calculated using RELION (Fig 4.3). These class averages were used for the 2D analysis of intact Elongator. Bad particles were excluded to yield a particle count of 9,962. The particles were then subjected to RELION 3D classification using the RCT model as a reference. Classes of inferior quality were discarded for a final particle count of 8,190. The final set of particles were used to refine the most highly 96  populated class from 3D classification using RELION’s auto-refine feature. At no point did we discriminate between ‘open’ and ‘closed’ lobe conformations of the complex. After postprocessing without map sharpening, a final resolution of 25 Å was calculated at the 0.143 FSC (Fourier Shell Correlation) criterion using the gold standard method240. Details on this process can be found in Figure 4.3. Similar procedures were used for the Elp123 reconstruction. 28,121 particles were extracted from 106 phase-flipped micrographs (Fig 4.7). Bad particles were discarded after 2D alignment and classification, and 16,362 particles were used for 3D classification with the RCT reconstruction of intact Elongator as an initial model. All particles segregated to classes resembling Elongator, and were used to refine the highest quality 3D model. As with full Elongator, we did not discriminate between the ‘open’ and ‘closed’ conformations of the complex. Details on this process can be found in Figure 4.7. For subunit localization 2D image analysis, all micrographs were binned twice to give a final pixel size of 4.7 Å/pixel. Individual particles were selected using EMAN Boxer189. Particle images were then aligned and classified in either SPIDER190 or RELION to generate the class averages. No significant differences between the performances of these two software suites were detected. Particles from class averages showing additional C-terminal MBP (Maltose Binding Protein) tag density were subjected to a second round of alignment and classification to further segregate particles with the additional MBP density. The same procedure of classification and subclassification was employed to determine the location of the anti-FLAG antibody bound to Elongator containing Elp1-FLAG.  97  4.2.6 Crosslinking coupled to mass spectrometry FLAG-purified Elongator (without glycerol gradient ultracentrifugation) were incubated with either 40, 80, 160, 240, 320, 480, or 640 μM of disuccinimidyl suberate (DSS) for 30 minutes are room temperature. This range of crosslinker concentrations were determined to generate both lightly and extensively crosslinked complexes by SDS-PAGE across two experiments with slightly different preparation methods until a satisfactory number of crosslinked peptides were detected. Reactions were quenched using 50 mM Tris-HCl pH 8.0 for 15 minutes at room temperature. Cross-linked samples were separated on 5-20% SDS-PAGE gels (BioRad, Hercules, CA) followed by Coomassie blue G-250 staining. The high molecular weight band corresponding to the cross-linked complex was excised and subsequently processed for mass spectrometry analysis as previously described224. Both experimental samples were digested with a combination of 10 ng/μL trypsin and 5 ng/μL Lys-C. Digested products were analyzed on a Q-Exactive MS instrument equipped with an Easy nano-LC 1000 liquid chromatography system (ThermoFisher Scientific, Waltham, MA). Digested peptides (~0.5 μg) were loaded onto 75 μm x 6 cm trap column that was packed with 10 μm, 120 Å ODS-AQ C18 resin (YMC, Kyoto, Japan) and connected through a microTee to a 75 μm x 10 cm analytical column packed with 1.8 μm, 120 Å UHPLC-XB-C18 resin (Welch Materials, Shanghai, China). Peptides were separated over a 100-min linear gradient from 100% buffer A (0.1% FA) to 30% buffer B (100% ACN,0.1% FA), then a 10-min gradient from 30% to 80% buffer B, reaching 100% buffer B in the next 1 min and maintaining at 100% buffer B for 2 min before returning to 100% buffer A in 3 min and ending with a 4-min 100% buffer A wash. The flow rate was 200 nL/min. 98  MS parameters: Top 20 most intense ions are selected for MS2 by HCD dissociation; R = 140,000 in full scan, R = 17,500 in HCD scan; AGC targets were 1e6 for the full scan, 5e4 for MS2; minimal signal threshold for MS2 = 4e4; +1, > +6 and unassigned precursors were excluded for identifying immune-precipitated products which +2 precursors were also excluded for cross-linking samples; normalized collision energy is 27 for HCD; peptide match is preferred. 4.2.7 Identification of cross-linking peptides and interacting proteins The MS data were analyzed using the pLink software tool178 to identify cross-linked peptides. The pLink search parameters were as follows: the protein database consisted of the sequences of six Elongator subunits and the proteases used to digest samples; maximum number of missed cleavages (excluding the cross-linking site) = 3; min peptide length = 4 amino acids, cysteine carbamidomethylation was set as fixed modification. pLink search results were filtered by requiring FDR ≤ 0.05 and a mass deviation ≤ 10-ppm of the observed precursor from either the mono-, the first, second, third, or fourth isotope of the matched candidate. Then the inter-linked peptides were further filtered by E-value < 0.0001 and spectra count ≥ 2. Results from this experiment are presented in Appendix A: Table A.3 4.2.8 Identification of co-purified proteins from Elongator subunit deletion strains Proteins co-purified with Elp4-FLAG from either elp1Δ, elp2Δ, and elp3Δ mutant strains were identified by searching the MS data against a Uniprot S. cerevisiae protein database using Prolucid241. The Prolucid search results were filtered using DTASelect 2242 by requiring ≤ 1% FDR at the peptide level. The protein FDR was < 6%. Technical duplicates were performed.    99  4.2.9 Yeast phenotypic assays Single colonies of each strain were grown in 5 ml Ura- medium at 30°C overnight with continuous shaking. For the zymocin growth curve assay, the overnight cultures were used to inoculate wells in a 48-well plate to a total volume of 200 µl per well, and an OD600 of 0.1 in technical triplicates. Conditions and strains in each well were randomized on the plate to account for non-uniform heating. Certain wells only contained Ura- media for background correction. +Zymocin media consists of 4:5 volume of Ura- media and 1:5 volume of concentrated filtrate isolated from a culture of Kluyveromyces lactis (NCYC 1368). The plates were incubated in the BioTek Synergy HTX microplate reader (BioTek, Winooski, VT) at 30°C with continuous orbital shaking for 16 hours. OD600 readings were taken every 10 minutes. For yeast plate assays, the cultures were grown to an OD600 ~ 0.5 and a five-fold serial dilution is performed. 5 µL of each dilution were spotted onto -Ura with and without 7 mM caffeine plates. Each plate was incubated for 2 days at 30°C before imaging. Three biological replicates were performed. 4.2.10 Elp2 loop mutant copurification Elongator was purified from yeast strains expressing both Elp2 loop mutants and Elp4-FLAG using the FLAG affinity purification procedure described above, with a few adjustments. Quantities of reagents and buffers used were adjusted for a starting culture volume of approximately 200 ml. Prior to incubation with α-FLAG M2 resin, the total protein concentration of the lysates were measured by Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA) and standardized across all strains analyzed. Glycerol gradient ultracentrifugation was not performed. Instead, FLAG elutions were analyzed by Western blot using α-HA antibody (Applied Biological Materials, Richmond, BC) and α-FLAG M2 antibody (Sigma-Aldrich, St. Louis, MO). 100  4.4.11 Construction of the multi-scale model of Elongator The EM map of intact Elongator was used as the basis for fitting. X-ray structures of the Elp1 C-terminus, Elp2, and Elp456 were obtained from PDB (5CQS, 4XFV, and 4A8J, respectively111,155,156), and homology structures of the Elp1 tandem WD40 domains, and the Elp3 radical SAM and GNAT domains were generated using Phyre2243. Initial manual rigid body fitting was performed in UCSF Chimera191 guided by the EM labeling experiments. Results from crosslinking coupled to mass spectrometry were used to provide additional restraints for further optimization of fitting of high-resolution structures within the map. 4.2.12 Overexpression and purification of recombinant Elp456 subcomplexes Full length yeast ELP4 and ELP5 were cloned into the pQLinkN vector and ELP6 was cloned into the pQLinkH vector containing an N-terminal 6xHis tag. The vectors were combined to make the pQLink-ELP4-ELP5-6xHis-ELP6 using ligation independent cloning methods244. The proteins were expressed in T7 Express competent E. coli (NEB, Ipswich, MA) at 18ºC for 20 hours using 1 mM Isopropyl β-D-1-thiogalactopyranoside (Gold BioTechnology, St. Louis, MO). E. coli pellets were harvested and sonicated in lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5% glycerol, 10 mM imidazole, 1 mM DTT, 1 mM PMSF). Lysates were cleared by centrifugation at 31,000x g and incubated with nickel-NTA resin (Thermo Fisher Scientific, Waltham, MA) for 1 hour at 4ºC then washed 6 times with wash buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5% glycerol, 50 mM imidazole). Bound proteins were eluted with elution buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5% glycerol, 250 mM imidazole). The eluted proteins were loaded onto a Superdex 200, 10/300 GL (GE Healthcare, Little Chalfont, UK) size exclusion chromatography column. The final buffer contains 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, and 1 mM MgCl2.  101  4.2.13 Malachite green ATPase assay Purified rElp456 was added to assay buffer (15mM Tris-HCl pH 7.5, 100mM NaCl, 1mM MgCl2) with or without 0.5mM ATP and incubated at 30°C for 30 minutes. Malachite green solution (Sigma-Aldrich, St. Louis, MO) was then added to the reaction and the absorbance at 620 nm was determined using the BioTek Synergy HTX microplate reader (BioTek, Winooski, VT). Absorbance readings were compared to a standard curve to calculate concentrations of free inorganic phosphate in each reaction. All assay conditions were done in triplicate. 4.2.14 Elongator and recombinant Elp456 mixing experiment FLAG eluates prepared from Elp1-FLAG as described above were incubated for 30 minutes on ice with purified recombinant Elp456 (rElp456) at a volume ratio of 3:1. The mixture, as well as rElp456 alone, were subjected to GraFix analysis as described above with identical buffer conditions.   4.2.15 Accession codes The final reconstruction for the intact Elongator complex and the Elp123 subcomplex was deposited into the EM Databank (entry number EMD-8239 and EMD-8291, respectively). 4.3 Results 4.3.1 Yeast Elongator adopts an asymmetric overall architecture We isolated the full Elongator complex from a S. cerevisiae strain encoding C-terminally FLAG-tagged Elp1 (Fig 4.1A). Densitometry analysis showed that the peak fraction obtained from an identical gradient run without crosslinker contains stoichiometric quantities of each of the six Elongator subunits (Table 4.2), confirming that this procedure preserved the integrity of Elongator. Negative stain EM analysis of purified Elongator showed elongated, bilobal particles  102   Figure 4.1. Elongator is a two-lobed asymmetric complex. Yeast Elongator was purified and analyzed by negative stain single particle electron microscopy. (A) Coomassie Blue stained SDS-PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C-terminally FLAG-tagged Elp1. (B) Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation (Lane 6 of Fig 1A). Circled particles correspond to intact Elongator. White scale bar corresponds to 50 nm. (C) 103  Representative 2D class averages of the full Elongator complex from a total of 17,074 particles. All class averages shown in this figure have side width of 500 Å. (D) Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure. (E) Six evenly-sized densities are arranged in one Elongator lobe. A projection of the Elp456 hexamer X-ray structure (PDB code: 4A8J) is shown for comparison. (F, G) Analysis of Elongator subunit localization using a C-terminal MBP tag. Class averages of untagged Elongator is shown for comparison. White arrowheads point towards additional densities in the tagged complex. (H) Mapping of Elp1 by incubating purified Elongator containing an Elp1 C-terminal FLAG tag with α-FLAG antibody. The additional antibody density is denoted by the white arrowhead. (I) Three dimensional reconstruction of the intact Elongator complex at 25 Å. (J) Schematic of the subunit locations based on 2D EM analysis. (K) Inset showing the cavity formed through the centre of the putative Elp456 density. Black scale bars in this figure correspond to 50 Å.  ‡Band fluorescence was obtained from the fraction corresponding to lane 5 of Figure 4.1A using the LI-COR Odyssey Imaging system Table 4.2. Band quantification of Coomassie blue stained SDS-PAGE gel of full Elongator Subunit Molecular Weight (Da) Molecular Weight Normalization factor Relative Band Fluorescence (700 nm)‡ Normalized Band Intensity Elp1 156,000 1 1 1 Elp2 89,000 1.75 0.604 1.057 Elp3 66,000 2.36 0.506 1.19 Elp4 51,000 3.06 0.449 1.37 Elp5 35,000 4.45 0.274 1.219 Elp6 31,000 5.03 0.234 1.177 104   Figure 4.2. Negative stain class averages of intact Elongator and the Elp123 subcomplex. This figure contains supporting information for the 2D negative stain analysis presented in Fig 4.1. (A) 17,074 particle images of Elongator purified from regular salt (150 mM NaCl) conditions were sorted and aligned into 50 classes. Class averages are sorted by distribution, with the classes containing the most particles displayed first. (B) Coomassie blue stained SDS-PAGE gel analysis of size exclusion chromatography fractions of the Elp123 subcomplex isolated using a high salt (300 mM NaCl) purification from yeast expressing FLAG-tagged Elp1. Lane I contains the concentrated FLAG eluate input. (C) Class averages calculated from 28,121 Elp123 subcomplex particle images. Class averages are sorted by distribution, with the classes containing the most particles displayed first.  105  with dimensions of approximately 300 Å x 200 Å (Fig 4.1B). Subsequent two-dimensional (2D) classification analysis revealed that Elongator adopts an overall shape resembling a moth, with two prominent triangular-shaped ‘wings’ connected at two corners by a small central density (Fig 4.1C panel 1).   The Elongator subunits have been proposed to associate into a symmetric complex, with two Elp123 subcomplexes connected to one another via the hexameric ring assembly formed by Elp456114. To our surprise, our 2D analysis showed that the majority of Elongator adopts an asymmetric architecture (Fig 4.2A). In particular, the most populated 2D class averages of intact Elongator show particles with a ring-shaped density perched on top of one of the two wings. Interestingly, no particles containing two ring-shaped densities were observed in our dataset (Fig 4.2A). In addition to the unique asymmetric architecture, our 2D analysis also revealed variability in the distance between the two lobes of the complex, with some particles showing a more ‘open’ conformation (Fig 4.1C panel 3, Fig 4.1D panel 1) and others a more ‘closed’ conformation (Fig 4.1C panel 4, Fig 4.1D panel 3). The overall dimensions and hexameric shape of the ring-shaped density resemble that of the projection view of the Elp456 crystal structure (Fig 4.1E). To determine if Elp456 constitutes this density, we isolated the Elp123 subcomplex from the Elp1-FLAG strain using a high salt purification strategy that dissociates Elp456 from the full Elongator complex239. Class averages obtained from 2D EM analysis of purified Elp123 show particles with both lobes lacking the ring-shaped density (Fig 4.1D, 4.2B-C). This result not only confirmed that Elp456 indeed binds to only one half of the Elp123 dimer in the intact complex but also showed that two Elp123 subcomplexes can dimerize in the absence of Elp456. 106  We next took an EM-based labeling approach to further investigate the organization of Elp123. We isolated Elongator containing C-terminal maltose binding protein (MBP) tagged Elp2 or Elp3 and localized the MBP labels by negative stain EM. We found that Elongator containing either MBP-tagged Elp2 or Elp3 displays additional densities at either the furthest corners or the posterior edge of the Elongator ‘wings’ (Fig 4.1F, G). Only one Elp3-MBP  density could be resolved from our 2D analysis. We attribute this to the class averaging procedure aligning one copy of the tag, while the variable relative position of the second tag resulted in its density being averaged down to background levels. To map the location of Elp1 C-terminus, we applied a similar EM-based approach using antibody-based labeling (Fig 4.1H). We observed an additional density corresponding to the antibody attaching to the tube-like density connecting the two Elongator lobes, a location consistent with recent findings that the Elp1 C-terminal domain mediates complex dimerization156. Collectively, these results indicate that Elp2 and Elp3 are positioned along the distal corners and posterior edges of the moth-shaped complex, respectively, while the Elp1 C-terminus likely forms the bridge between the two lobes of Elongator. The limited yield of our native complex purification have prevented us from preparing vitrified specimens and characterizing this complex at higher resolution by cryo-EM. We therefore determined a three-dimensional (3D) reconstruction of Elongator from our negative stained specimens (Fig 4.1I, 4.3). The resulting ~25 Å resolution structure confirmed that one of the two wing-shaped lobes houses an additional density with size and shape resembling the Elp456 hexamer. The plane of the Elp456 hexamer is angled approximately 30 degrees away from the complex plane (Fig 4.1I, top view) with the posterior edge closest to the Elp123 core complex. Aside from the putative Elp456 hexamer, each lobe can be divided into three main  107   Figure 4.3. 3D reconstruction of the Elongator complex. This figure contains supporting information to the 3D reconstruction of Elongator presented in Figure 4.1. (A) Random conical tilt (RCT) reconstruction generated from particles gathered at 60°/0° tilt angles. (B) RELION 3D classification results using the RCT model as a starting model. The Class 1 model was used for subsequent refinement. (C) RELION auto-refinement of the most populated 3D model using particles that were classified to classes 1, 3, 4, and 5 in the 3D classification step. (D) Gold-standard Fourier shell correlation curve indicating the resolution of the model. (E) Angular distribution representation of the input particles to the final model represented as a polar plot. θ and φ correspond to the rotation about the Y and Z axes, respectively. The radius of the plotted circles represent the number of particles in a particular orientation. Red orientations are ten times more populated than black ones. 108  densities arranged adjacent to each other. Based on our MBP-tagging experiments, we assigned the distal and central densities as Elp2 and Elp3, respectively (Fig 4.1J). Furthermore, the EM density map revealed a slender cavity, approximately 80 Å deep, that projects into the Elp123 lobe through the centre of the Elp456 hexamer (Fig 4.1K).  4.3.2 Subunit connectivity of holo-Elongator To gain further insights into the subunit connectivity of Elongator, we generated knockouts of three elp genes (elp1, elp2, or elp3) in a FLAG-tagged Elp4 background strain, and performing mass spectrometry analysis of anti-FLAG affinity purified samples from these three yeast strains to determine the effects of these mutations on complex formation (Fig 4.4A). We found that Elp5 and Elp6 co-purified with Elp4 in all three mutants, suggesting that the Elp456 ring can assemble independently of Elp123. Our analysis also showed that Elp1 is essential to the binding of Elp2 and Elp3 to Elp456, while Elp2 deletion has no effects on the association of Elp1 and Elp3 to Elp456. Finally, Elp3 deletion severely disrupts Elp2 but not Elp1 binding to Elp4. Results from these studies are consistent with the observed peripheral location of Elp2 and in addition indicated that Elp1 not only mediates complex dimerization but also serves as the major scaffold for other Elongator subunits to assemble.  To gain molecular insights into subunit organization, we subjected purified Elongator to crosslinking coupled to mass spectrometry (CXMS) analysis. We incubated purified Elongator with the crosslinker disuccinimidyl suberate (DSS) and analyzed the crosslinked peptides by mass spectrometry to identify lysines in close proximity to each other (Fig 4.4B-D). We found that 24 out of 25 crosslinked residues observable in available high-resolution structures of Elongator subunits were within the 30 Å maximum Cα-Cα distance allowable by DSS245 (Table 4.3).  109   Figure 4.4. Mass spectrometry analysis of Elongator. In order to analyze the subunit composition of the complex, several experiments using mass spectrometry were performed. (A) Number of peptides detected of each Elongator subunit purified through Elp4-FLAG pulldown from yeast lacking ELP1, ELP2, or ELP3 as a fraction of the number of Elp4 peptides. Individual data points are represented as circles, while the averages are represented as bars. (B,C) Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate (DSS) and analyzed by mass spectrometry. Schematic representation of the major crosslinking connectivity of the complex is shown. (D) Detailed locations of crosslinked residues plotted onto a domain representation of Elongator. Lysines from two residues found crosslinked together are denoted by black lines. 110  Table 4.3. Validation of crosslinks detected in Elongator CXMS experiments  Subunit 1 Residue 1 Subunit 2 Residue 2 CA-CA Distance Major fold* PDBID Elp1 967 Elp1 967 109.3 TPR(A)-TPR(B)‡ 5CQS Elp1 967 Elp1 989 16.2 TPR-TPR   Elp1 989 Elp1 1020 11  “   Elp1 1030 Elp1 1052 13.2  “   Elp1 1052 Elp1 1074 8.5  “   Elp1 1270 Elp1 1304 15.6 3HB-3HB   Elp2 25 Elp2 28 5.7 7βP(1)-7βP(1) 4XFV  Elp2 49 Elp2 768 18.5 7βP(1)-7βP(2)   Elp2 460 Elp2 553 10.4 7βP(2)-7βP(2)   Elp2 460 Elp2 599 18.8  “   Elp2 619 Elp2 686 16.7  “   Elp2 629 Elp2 647 7.8  “   Elp2 629 Elp2 650 5.6  “   Elp2 647 Elp2 680 14.5  “   Elp2 650 Elp2 680 12.2  “   Elp2 650 Elp2 699 16.6  “   Elp2 677 Elp2 699 5.3  “   Elp2 677 Elp2 702 5.7  “   Elp2 680 Elp2 699 8.8  “   Elp5 47 Elp5 100 14.8 RecA-RecA 4A8J Elp5 211 Elp4 375 22.1  “   Elp6 225 Elp4 399 14.2  “   Elp6 230 Elp4 399 14.4  “   Elp6 230 Elp6 265 13.3  “   Elp6 230 Elp6 269 13.3  “    * TPR: Tetracotripeptide repeat; 3HB: 3-helix bundle; 7βP(1)/ 7βP(2): 7-beta propeller, N- /C-terminal domain, RecA: RecA fold ‡ A and B refers to the two chains of the Elp1 dimer   The Elp1 N-terminal domain, Elp2, Elp3, and Elp4 contained the highest number of crosslinks, suggesting that these subunits likely form the primary interaction interface of the complex (Fig 4.4B-D). Elp3 was found crosslinked to every unique subunit of Elongator. This observation is consistent with the central location of this subunit deduced from our EM-based labeling analysis. Furthermore, by mapping the crosslinked residues onto the crystal structure of 111  the core Elp456 hexamer155, we found that the β sheet-rich face of the Elp456 housing the N and C termini of Elp5 and Elp6 is likely oriented towards the Elp123 core complex.  4.3.3 Conserved loop regions of Elp2 are crucial to Elongator function To further validate our CXMS data, we probed a series of four loop regions in Elp2 that were found to be crosslinked to other Elongator subunits. We hypothesized that these loops are important for Elp2’s role in Elongator function, possibly in stabilizing major interaction interfaces. We introduced alanine mutations to the conserved residues within four loops (Fig 4.5A) and examined their effects on Elongator function. We found that mutations in loops 1, 3, and 4 of Elp2 cause impaired growth on plates containing caffeine (Fig 4.5B). These mutants are also resistant to the Kluyveromyces lactis toxin zymocin, which specifically cleaves tRNA harboring U34 (wobble position) modification (Fig 4.5C)152. Both these phenotypes are consistent with impaired Elongator function148,246.  We next evaluated the association of these Elp2 mutants to other Elongator subunits. We purified Elongator from strains expressing the Elp2 loop mutants and Elp4-FLAG using anti-FLAG affinity chromatography, and probed the eluted proteins by Western blot (Fig 4.5D). We found that the Elp2 loop 2 mutations did not affect co-purification with Elp4-FLAG. In contrast, the loop 1 mutation results in substantially reduced level of Elp2 co-purified with Elp4 (Fig 4.5D). The loop 3 and 4 mutations led to a mild reduction in co-purification, suggesting that these loops mediate Elp2 function aside from complex assembly. These results confirm that the highly crosslinked, conserved residues in the loop 1, loop 3, and loop 4 regions of Elp2 are critical for Elongator function.    112   Figure 4.5. Conserved Elp2 loops are important for Elongator function. Loop regions of Elp2 enriched for crosslinks in CXMS analysis were mutated and analyzed for Elongator defects. (A) Schematic of the conserved residues within four Elp2 loops that were mutated to alanine within an ELP2 plasmid. (B) Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto –Ura media containing caffeine. The pRS416 vector is used for these experiments. (C) Growth curve analysis of loop mutants in –Ura media with and without K. lactis toxin zymocin. Shaded regions correspond to the SEM at each time point. N=3. (D) Lysates of Elp4-FLAG-containing yeast expressing the Elp2 loop mutants were 113  incubated with α-FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α-HA and α-FLAG antibody  4.3.4 Model of yeast Elongator architecture In order to visualize the overall architecture of Elongator, we took a multiscale modeling approach that involves integrating: (i) EM mapping data, (ii) the subunit crosslinking patterns, (iii) the available crystal structures of Elp1 C-terminal domain156, Elp2111, and the core Elp456 hexamer155, and (iv) homology models of the Elp1 N-terminal WD-40 and the Elp3 HAT and radical SAM domains to derive a ‘best fit’ multiscale model representing the approximate overall architecture of Elongator (Fig 4.6A,B).  Our model shows that the two Elp1 subunits form a central scaffold and dimerize at their C-terminal regions, linking the two wing-shaped lobes. Elp1, Elp2, and Elp3 constitute the three adjoining densities forming the central framework of the complex. The lone Elp456 ring is anchored to one of the two Elp123 lobes. The 80 Å deep cavity spanning the centre of Elp456 contacts the Elp3 HAT and radical SAM domains extensively (Fig 4.6B). Since residues lining the centre of the hexamer were shown to be important for tRNA binding155, and that the cavity comfortably fits a tRNA molecule, we speculate that this pocket may serve as the tRNA binding site.  Our model is consistent with our CXMS results, with direct access between crosslinked lysine pairs. However, the distance between several Elp2-Elp1N crosslinks exceed the predicted DSS length. This is likely due to a combination of imperfect homology modeling of the Elp1 N-terminal WD40 domains and potential conformational flexibility of the Elp123 lobe. Flexibility  114     Figure 4.6. Model of Elongator subunit organization and Elp456 association. (A) CXMS and EM data, combined with the X-ray structures of Elp1 C-terminus (5CQS156), Elp2 (4XFV111), Elp456 (4A8J155), and a homology model of the Elp1 tandem WD-40 domains, were used to estimate the relative positions of each subunit to generate a multi-scale model of Elongator subunit organization. (B) Side view of the Elongator architectural model highlighting the cavity (red outline) spanning through the centre of the Elp456 hexamer. (C) Comparison of the Elp123 conformations of the intact Elongator complex (gold) and the Elp123 subcomplex (cyan). The Elp456-unloaded lobe of Elongator and one lobe of Elp123 were aligned.  115  within macromolecules often result in the violation of the allowable intersubunit crosslinking length176,247, and movement within the Elp123 lobe may transiently shorten the distance between Elp1 and Elp2. A key requirement of our model is that the two Elp123 lobes have to be arranged in an asymmetric fashion in order to fit within the molecular envelope of the EM density map. A major implication of this is that binding of Elp456 induces a conformational change in the Elp123 lobe. In order to test this hypothesis, we generated a three dimensional reconstruction of the Elp123 subcomplex alone and overlaid it onto the EM density map of full Elongator (Fig 4.6C, 4.7). Strikingly, Elp123 alone adopts a more extended conformation of the two Elp123 lobes (Fig 4.6C, front view, Fig 4.7E) and a different relative rotational state relative to the putative Elp1 C-terminal bridge (Fig 4.6C, top view). This finding leads us to propose that the Elp456 loading to one Elp123 lobe induces a conformational change.  4.3.5 Elp123 can accommodate two Elp456 subcomplexes We investigated the possibility that binding of one Elp456 hexameric ring prevents loading of a second ring in the opposite lobe. In order to establish that Elongator only contains one Elp456 ring in vivo, we purified the complex using a low salt buffer. Since high salt dissociates Elp456 from Elongator, we hypothesized that low salt conditions may preserve Elongator containing two Elp456 rings. Despite a less efficient purification, class averages of these complexes largely show identical, asymmetric Elongator observed previously (Fig 4.8A,B, 4.9A). However, several well populated class averages show an increased density in both lobes that may correspond to a second Elp456 ring (Fig 4.8B, class average indicated by a white triangle).  116   Figure 4.7. 3D reconstruction of the Elp123 subcomplex.  This figure contains supporting information to Figure 4.6. (A) Three dimensional reconstruction of the Elp123 subcomplex. (B) Elp123 reconstruction at a lower threshold value, revealing noise artifacts and the connectivity between the two halves of the complex. (C) Angular distribution representation of the input particles to the final model represented as a polar plot. θ and φ correspond to the rotation about the Y and Z axes, respectively. The radius of the plotted circles represent the number of particles in a particular orientation. Red orientations are ten times more populated than black ones. (D) Gold-standard Fourier shell correlation curve indicating the resolution of the Elp123 reconstruction. (E) Particles of Elongator and Elp123 in the front-facing orientation were isolated from their respective datasets and analyzed by alignment and class averaging. The widths of the 117  complexes in the resulting class averages were plotted in bins of 3.5 Å. (F) Comparison of Elongator and Elp123 top views illustrating conformational differences.  If Elongator can accommodate two Elp456 rings, we hypothesized that the formation of a double-loaded Elongator complex can be forced in vitro through mass action. We expressed and purified recombinant, catalytically active Elp456 subcomplexes from E. coli (Fig 4.8C, 4.9B,C). We then incubated an excess of recombinant Elp456 (rElp456) with intact Elongator purified from yeast in buffer with the regular 150 mM NaCl concentration (Fig 4.8C, 4.9C). We subjected the mixture, as well as rElp456 alone, to glycerol gradient ultracentrifugation and analyzed the fractions by Coomassie-stained SDS-PAGE and negative stain EM (Fig 4.8D-F, 4.9). As expected, glycerol gradient ultracentrifugation of rElp456 alone shows that the smaller subcomplex did not penetrate past the first three fractions (Fig 4.8D). Strikingly, much less rElp456 accumulated at the top of the gradient containing both Elongator and rElp456 (Fig 4.8E). Band density analysis of the peak Elongator fraction revealed that there is approximately twice as much Elp4, Elp5, and Elp6 compared to Elp1 (Table 4.3). Interestingly, Elp3 seems to also be enriched relative to Elp1 and Elp2 in this fraction. Negative stain EM analysis showed mostly class averages corresponding to Elongator containing two Elp456 rings (Fig 4.8F, 4.9D). These results suggest that two copies of Elp456 subcomplexes can bind to Elongator even in moderate salt conditions. 4.4 Discussion This study presented the first comprehensive structural view of the intact Elongator complex. Previous research have shown that Elongator is inherently dimeric and consists of two copies of six unique protein subunits155. Glatt et al proposed that Elp456 acts as a centrally  118   Figure 4.8: Elongator can accommodate two copies of the Elp456 subcomplex. (A) Silver-stained SDS-PAGE of Elongator glycerol gradient fractions purified using a low salt (75 mM NaCl) buffer. The fraction containing the most Elp4, Elp5, and Elp6 is indicated with * and the corresponding fraction from a glutaraldehyde-containing glycerol gradient is used for EM analysis. (B) Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations (150 mM and 75 mM NaCl, respectively). Densities resembling a second copy of Elp456 is indicated by white triangles. (C) Scheme of the Elongator and rElp456 mixing experiment. Recombinant Elp456 subcomplex (rElp456) was purified from E. coli and mixed with intact yeast Elongator in regular salt conditions. The mixture, as well as rElp456 alone, 119  were then subjected to glycerol gradient ultracentrifugation. (D, E) Coomassie-stained SDS-PAGE of rElp456 alone (D) or rElp456 with intact Elongator (E). The fraction containing the most intact Elongator complex is indicated with *, and the corresponding fraction from a glutaraldehyde-containing glycerol is used for EM analysis. (F) Comparison of class averages of natively purified Elongator with and without rElp456 incubation. Densities resembling a second copy of Elp456 is indicated by white triangles.     Table 4.4. Band quantification of Coomassie blue stained SDS-PAGE gel of full Elongator incubated with an excess of recombinant Elp456 Subunit Molecular Weight (Da) Molecular Weight Normalization factor Relative Band Fluorescence (700 nm)‡ Normalized Band Intensity Elp1 156,000 1.00 1 1 Elp2 89,000 1.75 0.584 1.02 Elp3 66,000 2.36 0.715 1.69 Elp4 51,000 3.06 0.621 1.9 Elp5 35,000 4.46 0.400 1.78 His-Elp6 33,800 4.62 0.149 0.69 Elp6 31,000 5.03 0.328 1.65 ‡Band fluorescence was obtained from the lane marked with * in Figure 4.8 using the LI-COR Odyssey Imaging system 120     Figure 4.9. Investigating loading two copies of Elp456 on the Elongator complex. This figure contains supporting information to Figure 4.8. (A) Negative stain EM analysis of Elongator in low salt conditions. A representative micrograph segment of this fraction is shown. The scale bar corresponds to 50 nm. The right panel shows a gallery of 2D class averages of this sample. The box size of each class average corresponds to ~60 nm. (B) The recombinant Elp456 (rElp456) subcomplex was purified from E. coli and its ATPase activity assayed using Malachite green phosphate assay. Error bars correspond to standard error of mean (N=3). (C) Coomassie-stained SDS-PAGE of semi-purified FLAG elutions of intact Elongator and purified recombinant Elp456 subcomplex. The two complexes were incubated together prior to glycerol gradient ultracentrifugation. (D) Negative stain EM analysis of Elongator incubated with rElp456. A representative micrograph segment of this fraction is shown. The scale bar 121  corresponds to 50 nm. The right panel shows a gallery of 2D class averages of this sample. The box size of each class average corresponds to ~60 nm.  located bridge between the two Elp123 subcomplexes. However, this model was challenged by the recent crystallographic analysis of the Elp1 C-terminal domain, which revealed that this domain self-dimerizes and facilitates Elongator dimerization156. Our results clearly show that the Elp456 heterohexamer is anchored to only one Elp123 lobe, and distal to the dimerization interface. The 3D reconstruction of Elongator show a long and slender bridging density, consistent with the alpha-helical repeats of the C-terminal TPR/IKBKAP (TetratricoPeptide Repeat/Inhibitor of Kappa light polypeptide gene enhancer in B-cells Kinase complex Associated Protein) dimerization domain156. Therefore, we conclude that Elongator is an asymmetric ‘dimer of multimers’ likely connected by the Elp1 C-terminal region. We also observed that Elp123 changes conformation upon Elp456 binding (Fig 4.6C). Interestingly, the family of RecA-like ATPases that Elp456 belongs to contain many examples of molecular motors, such as helicases and transporters that utilize NTPs to conduct mechanical work157. The conformational changes of Elp123 could potentially be induced by Elp456’s catalytic activity. The mechanism limiting Elp456 association to only one Elp123 lobe is not known. We observed that the majority of front views of the complex contain only one copy of the Elp456 ring. A minor population of particles generated a class average with no discernable ring densities essentially identical to the class averages of obtained for purified the Elp123 subcomplex (Fig 4.2A,C). Furthermore, none of the class averages we obtained for full Elongator show two Elp456 rings associated with both Elp123 lobes. However, we show that incubating purified Elongator with an excess of recombinant Elp456 subcomplexes result in the association of the 122  heterohexameric rings to both Elp123 lobes (Fig 4.8F). This experiment indicates that there is no direct mechanism preventing double loading of Elp456 subcomplexes to Elp123. However, we also show that complexes purified from yeast, even under low salt conditions, mostly only contains one Elp456 ring (Fig 4.8B). Elongator stoichiometry may therefore be regulated at the level of expression, as Elp1, Elp2, and Elp3 have been shown to be more abundant in the cell than Elp4, Elp5, and Elp6248.  Our study provides the first insight into the structure of the holo-Elongator complex. In order to improve upon our architectural model, high-resolution structural data on the Elp1 N-terminal domain and Elp3 as well as the intact complex will be required. Whether Elongator contains two copies of the Elp456 heterohexamer in vivo remains an open question. We and other groups have shown that purified Elongator is most stable in conditions that result in a 1:1:1:1:1:1 subunit stoichiometry148,155, but whether the 1:1:1:2:2:2 complex is present and functional in vivo still needs to be addressed in future studies.         123  Chapter 5: Conclusions and future directions Eukaryotes employ a myriad of different mechanisms to regulate their gene expression. At the core of these mechanisms is transcription regulation through chromatin structure. Upon closer inspection, the seemingly straightforward action of physically blocking transcription machinery from DNA using nucleosomes is controlled by a highly complex interplay between hundreds of different factors shaping the chromatin landscape. Acetylation of histone tails is one axis of chromatin regulation, being the first post-translational modification of histones shown to stimulate transcription. Since the discovery of these histone marks, immense effort was dedicated to identify the enzymes responsible for their deposition. Decades of painstaking biochemical and genetic work—much of it done in budding yeast S. cerevisiae—revealed that these enzymes are often part of large, modular, and multifunctional complexes. There is a wealth of information available on the structure and function of these complexes, much of it centred on either individual subunits or modules. However, very little is known how each complex’s subunits function and cooperate within the context of these large molecular machines.  This dissertation aims to address this gap in knowledge by carefully examining the molecular architecture of the subunits within lysine acetyltransferase complexes. Determining the spatial organization of the subunits and modules in these complexes is a crucial first step towards understanding their precise mechanisms of action. My dissertation aims this architecture-centric paradigm to several different acetyltransferase complexes, using various techniques that probe the arrangement and connectivity of their subunits. I chose to focus on three enigmatic complexes: SAGA, NuA4, and Elongator.  In Chapter 2 of this dissertation, I delved into the molecular architecture of the yeast Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex. SAGA was the first KAT complex to be 124  characterized, and also holds the distinction of being the largest. In 2004, Wu et al utilized single particle electron microscopy to study the overall structure of the complex, as well as localizing several of its subunits181. I sought to expand upon this work, as new SAGA subunits and new techniques in analyzing large complexes have since been characterized. I discovered that using glutaraldehyde crosslinker stabilized the complex and drastically improved the quality of electron microscopy analysis of the complex. These improved images revealed that SAGA adopts three distinct conformations, one of which is dependent upon the presence of the deubiquitination module. This work also embarked upon an extensive subunit localization effort, using GFP tagging, subunit mutations, and antibody labeling to determine the approximate locations of subunits and modules in the complex. I compounded this information with a detailed subunit connectivity analysis using crosslinking coupled to  mass spectrometry to generate a model of SAGA architecture. This model placed the acetyltransferase module of the complex at the highly flexible tail of the complex, with the deubiquitination module in close proximity. These two modules, as well as several other subunits containing chromatin reader domains, form a putative nucleosome-interacting cleft. Their geometry suggests that it is possible for SAGA to apply both its catalytic activities simultaneously to the same nucleosome, and the region’s flexibility may accommodate for variability in the chromatin template.   Chapter 3 of this dissertation seeks to elucidate the architecture of the yeast NuA4 complex. NuA4 is the only essential KAT in S. cerevisiae, and, peculiarly, shares 12 out of its 13 subunits with other chromatin-modifying complexes. I embarked upon this work in response to a study by Chittuluru et al characterizing NuA4 using single particle electron microscopy200. The reported structure was strikingly similar to one region of SAGA even though the two complexes only share one subunit, and I believed that the reported 3D reconstruction was too small to 125  accommodate all the subunits of NuA4. Using the same strategy employed for SAGA, I studied the molecular architecture of this complex. As suspected, EM studies of NuA4 purified using an alternative buffer revealed two strong additional densities that were not reported previously. Subunit mapping efforts found that these additional densities consist of the shared SWR1C and Eaf5/7/3 modules, and both contain chromatin-interacting domains. The arrangement of these two densities relative to the catalytic acetyltransferase module is reminiscent of SAGA’s putative nucleosome binding cleft, and suggests that these domains can engage a nucleosome simultaneously. Through a collaboration with Dr. Jacques Côté, I also began initial structural characterization of the TIP60 complex, which seems to be an amalgamation of yeast NuA4 and SWR1 complexes133. The EM analysis of this complex recapitulated this observation, as the TIP60 structure quite strongly resembles that of yeast NuA4 stacked on SWR1. This study of NuA4 structure shows that the complex is a series of modules anchored together, as opposed to one globular complex.  Chapter 4 represents a departure from the preceding two chapters, as it focused on a complex that has not been shown to be a bona fide histone acetyltransferase: Elongator. I initiated structural analysis of this complex due to the presence of a lysine acetyltransferase domain of the same family as SAGA’s KAT subunit Gcn5. To date, however, there is no unequivocal evidence that Elongator acetylates histones in vivo. Instead, it participates in wobble base pair modification of tRNAs114, possibly adapting the KAT domain activity to participate in this pathway81. Despite the catalytic domain similarity with lysine acetyltransferase complexes, our structural analysis shows a very different complex to either SAGA or NuA4. Importantly, Elongator arranges into an asymmetric dimer, with two copies of Elp1, Elp2, and Elp3 forming a moth-shaped scaffold with Elp456 assembling as a heterohexameric ring associating with only 126  one wing. Although this asymmetric complex is the most stable form in vitro, Elongator can accommodate two copies of the Elp456 ring when incubated with an excess of the heterohexamer. These findings suggest that the Elongator asymmetry is stoichiometrically controlled, and may allow the catalytic Elp3 to process two different substrates in the same complex. This study represents the first effort to characterize the structure of the intact Elongator complex. 5.1 Module sharing between KATs and other chromatin-modifying complexes  My research into these three complexes brought to light several intriguing observations. First is the modular architecture of SAGA and NuA4. Most of the subunits in these complexes can be categorized into functionally distinct modules, and oftentimes these modules can be dissociated through mutation of one anchoring subunit. My preliminary experiments analyzing the unanchored KAT and DUB modules of SAGA show that they can independently be recruited to the nucleus. Most of the subunits in SAGA and NuA4 are shared with many other chromatin-related complexes, with only the Spt and Eaf1 subunits being unique to their respective complexes. Are these complexes dynamically sharing these subunits between each other? This raises the possibility that competition between complexes for their respective subunits may serve as a layer of regulation for these chromatin-modifying complexes. If this were the case, changes in the ratio of these complexes would alter the transcriptional regimes of the genes that they target, generating an intricate feedback loop. Research into the stoichiometric regulation of chromatin-modifying complexes may yield valuable insights into the chromatin modification network.  Experiments delving into this problem would likely begin with the initial hypothesis that chromatin modifying complexes compete for shared subunits. To use SAGA and NuA4 sharing 127  of Tra1 as an example, I would first carefully determine the stoichiometry of Tra1 relative to SAGA and NuA4 subunits. If it is not limiting, then the promoter can be mutated to artificially reduce expression of Tra1. The relative levels of intact SAGA or NuA4 can then be measured by affinity pulldowns of unique subunits and probing for the amount of Tra1 being co-precipitated. The same experiment would be repeated with a mutation that prevents assembly of either complex. If SAGA and NuA4 compete for Tra1, disrupting one complex would increase Tra1 association with the other. One intriguing experiment using this system involves subjecting Tra1-limited yeast to heat shock or nutrient stress; both conditions require SAGA to upregulate the appropriate stress response genes115. Would the relative amount of Tra1 association with SAGA and NuA4 shift in favour of SAGA? If subunit and module sharing is a form of regulation, I would hypothesize that mechanisms exist to shift limiting subunits to the appropriate complex, either pre- or post-assembly, given changing environmental conditions. Another approach would be to introduce mutations that specifically disrupt the association of shared subunits with one complex but not the other. If subunit sharing is a limiting step, these mutations should increase the activity of the complex that is still able to associate with the mutated subunit.  Another aspect of the module sharing hypothesis is whether complexes dynamically switch shared subunits, or if stoichiometric effects only take place pre-assembly. I would first address this question in vitro using an enzymatic protein fragment complementation approach249. One fragment would tag Tra1 from the NuA4 purification strain, and the other fragment would tag a SAGA subunit from the SAGA purification strain. Although manipulation of the massive Tra1 gene carries its own set of challenges, plasmids expressing the subunit have been successfully constructed previously130. The two purified proteins would be mixed and monitored for enzymatic complementation, which would indicate dynamic switching of Tra1. If this were 128  the case, an excess of unlabeled Tra1 would then diminish the enzymatic complementation signal. The same experiments can be repeated in vivo using split GFP experiments250. Tra1 and another NuA4 subunit would carry the complementary GFP fragments in an ada1Δ strain with limiting Tra1. A plasmid with inducible expression would code for the Ada1 subunit. Induction of Ada1 expression rapidly followed by a cycloheximide block of further translation would then cause the GFP signal between Tra1 and the NuA4 subunit to diminish if SAGA competes for Tra1 through dynamic module switching.   These proposed experiments would show whether or not complexes compete for subunits. Functional effects would require additional approaches such as transcriptome analysis of mutants with different expression levels of shared subunits. Since Tra1 is the activator targeting module of both SAGA and NuA4, chromatin immunoprecipitation coupled with sequencing targeting Gcn5 and Esa1 with varying levels of Tra1 would reveal any changes in chromatin association of each complex. Module sharing may reveal another facet of chromatin regulation, and shift the perception of chromatin-modifying complexes from ever-present regulators to valuable limited resources that the cells mobilize as necessary. 5.2 The role of Elongator in the cell  The shared modularity of SAGA and NuA4 does not extend to Elongator, however. Although I did not explicitly study the in vivo role of the complex, my research has provided several insights into aspects of Elongator distinct from chromatin modifying complexes. Disruption of the different modules in SAGA and NuA4 result in heterogeneous phenotypes, while Elongator subunit disruptions all result in nearly identical phenotypes79. Secondly, SAGA and NuA4 are very stable in high salt conditions, whereas Elongator is not. This, compounded with the observation that yeast Elongator localizes to the cytoplasm strongly suggest that these 129  complexes do not function in the same environment25. Finally, Elongator does not contain specific chromatin-interacting domains. Although WD40 domains, present in Elp1 and Elp2, can interact with histones, it is a general protein-protein interaction mediator that likely facilitates complex assembly instead. Furthermore, a recent crystal structure of an archaeal Elp3 revealed that the region of the KAT domain used to specifically interact with histones is inaccessible81. The extent of Elongator activity aside from wobble base pair modification is unclear, however.  Elongator is not an area of intense study in part because of the controversial nature of its function. Few would argue against Elongator’s role as a mediator of tRNA wobble base pair modification. As mentioned above, the observations I have made in my research do argue against a chromatin-centric role of the complex. However, the hotly debated shift from transcriptional to translational regulator resulted in a very defensive field wary of any suggestion that Elongator may function in other pathways. At the root of the problem is the pleiotropy of elp mutant phenotypes. Translation, like transcription, is a bottleneck that every protein-mediated cellular pathway must pass. Therefore any translational defect can result in a huge range of phenotypes, and any interesting effects of elp mutants face an uphill battle to be considered as a result of an independent pathway. I believe that any research into other Elongator function requires some means of divorcing the complex from wobble base pair modification. The first method bypasses wobble base pair modification by enriching for tRNAs themselves, and has already been conducted by the Byström group to show that elp mutant phenotypes are rescued by tRNA overexpression79. Unfortunately, no more research has been done on Elongator function in these tRNA-enriched strains. Transcriptome analysis of wild type strains, elp mutants, and elp mutants with overexpressed tRNAs might help to tease out any transcriptional effects of Elongator independently of wobble base pair modification. However, any phenotypes uncovered using 130  these strains can be dismissed as imperfect elp mutant complementation of tRNA overexpression. A second, more rigorous method involves finding a surrogate for wobble base pair modification. Elp3 is a very well conserved protein found in every single kingdom of life251. Overall tRNA structure and codon-anticodon interactions are likely more conserved than protein interfaces252. Therefore, there is probably an Elp3 homologue that can rescue tRNA wobble base pair modification while being unable to interact with S. cerevisiae-specific pathways. Using either tRNA overexpression or surrogate wobble base pair modifying enzymes would clear the path for research into other Elongator-mediated pathways.  Finally, another important avenue of research is the role of Elongator in mammals. In human cells, Elongator co-immunoprecipitated with the coding regions of genes and localizes to both the cytoplasm and the nucleus253,254. These observations suggest that Elongator can play some transcriptional role in mammals. Furthermore, truncations in human Elp1 (IKBKAP)  results in familial dysautonomia, a rare neurological disease that damage the autonomic and sensory nervous systems237. Studying Elongator in humans is therefore crucial in understanding both its basic biological function and in the context of biomedical research. 5.3 High resolution structural studies of KAT complexes  This dissertation presented the first steps towards comprehensive structural characterization of the SAGA, NuA4, and Elongator complexes. Although determining the overall spatial organization of each complex’s subunits provided insights into how these complexes engage their substrates, the low resolution of these analyses means that the precise folds that the subunits adopt in the context of the complex remains unknown. High resolution structures of these complexes is the logical future direction for research into their architecture.  131   Throughout my research, however, one problem dominated any endeavour to determine high resolution structure determination: yield. Negative stain analysis uses a layer of carbon that can be utilized to concentrate proteins via adsorption. However, my experience with all three complexes that I worked on is that carbon surfaces tend to induce preferred orientations. 3D reconstruction using electron microscopy requires a variety of orientations to ensure that there is no anisotropy in the final model. In contrast to negative stain, cryo-EM typically freezes an unsupported layer of vitreous ice (although technically it is supported by holey carbon, only hole regions devoid of carbon are imaged). This precludes using adsorption to concentrate the sample on the grid, therefore requiring a much higher protein concentration. Although cryo-EM can also use grids with a carbon layer, the induced preferred orientation and additional noise severely limits the quality of the dataset. Key to any future high resolution studies, then, is a much improved purification yield.  Several approaches can be used to improve the final amount of purified protein. The most obvious method, if somewhat inelegant, is to upscale the purification. Out of the complexes I worked with, Elongator has the highest purification yields (low micrograms). 32 L of heavily overgrown yeast (approximately 200 g of pellet) was barely enough for cryo-EM sample preparation. Therefore any serious future efforts would likely employ fermenters to grow over 100 L of culture, and the entire process likely has to be repeated to optimize freezing conditions. A second approach is recombinant overexpression of every subunit within the complex. Insect cell expression has resulted in spectacular protein complex preparations: The Barford group overexpressed all 13 subunits of the S. cerevisiae anaphase promoting complex in insect cells and determined an 11 Å resolution structure of the complex255. One caveat to this technique is that recombinant complexes lack the post-translational modifications of natively purified 132  proteins. As observed with the Elongator complex accommodating two Elp456 hexamers, in vivo stoichiometry may also control complex composition. Rigorous experiments must be performed to ensure that the recombinant complexes reflect their in vivo forms. These caveats aside, and although cloning and expression optimization of so many subunits is a substantial challenge, recombinant overexpression is a promising avenue for the study of large complexes.   As with all structural biology, the quality of the sample dictates the quality of the final structure. Any cryo-EM endeavour faces monumental challenges in preparing purified samples of sufficient quality and quantity for data collection. Oftentimes, the plunge-freezing process can severely detract from the data quality of even the most well-prepared protein samples. Once these challenges are surmounted, however, a high resolution cryo-EM structure is often within reach. In the past five years, once-impenetrable barriers in the field fell in rapid succession in a phase dubbed the ‘resolution revolution’ brought about by a new generation of direct-electron detectors256. These detectors have a higher quantum efficiency than film or charge-coupled device (CCD) cameras, allowing for increased signal at higher resolutions257. Electron beam induced motion was circumvented by collecting data as movies, and aligning individual frames258. Contrast problems due to small protein complexes were addressed by Volta phase plate technology and aggressive electron dose coupled to dose fractionation259,260. Structural flexibility can be accounted for through classification and masking approaches222,261. Even the intense computational requirements have largely been allayed by GPU (graphics processing unit) computing262. High resolution cryo-EM will generate structures that drastically improves our understanding of KAT complexes. 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Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence inter ADA1_Q12060(116)-ADA3_P32494(416)    2 2 3.62E-09 ELSNELELVFSPSAASLEKSNTNHHHSLVR(19)-DDNDKSELQSSK(5) inter ADA1_Q12060(167)-ADA3_P32494(563)    3  2.31E-05 NGNESSWGFGNGSNNPNNKLK(19)-ELQGTLKQVTK(7) inter ADA1_Q12060(234)-SPT20_P50875(479)    2  1.85E-05 IPIVTNPESLKR(11)-LKFIEQWR(2) inter ADA1_Q12060(234)-TRA1_P38811(2781)     3 9.91E-06 VADWNSDRDALEQSVKSVMDVPTPR(16)-IPIVTNPESLKR(11) inter ADA1_Q12060(237)-SPT20_P50875(479) 1   3 3 8.69E-09 LKFIEQWR(2)-VKSNNLK(2) inter ADA2_Q02336(101)-GCN5_Q03330(207)    3 9 8.82E-10 NTSNIKYFLTYADNYAIGYFK(6)-GKEEVKEHYLK(6) inter ADA2_Q02336(112)-ADA3_P32494(225) 1 1  8 12 6.08E-17 YYLESKYYPIPDITQNIHVPQDEFLEQR(6)-NPKSEFVVSQTLPR(3) inter ADA2_Q02336(299)-ADA3_P32494(366) 1   4 5 2.75E-06 SNGLTTLEAGLKYER(12)-KPSPYSNDASTILPK(1) inter ADA2_Q02336(313)-ADA3_P32494(366)     3 2.84E-10 ISSFEKFGASTAASLSEGNSR(6)-KPSPYSNDASTILPK(1) inter ADA3_P32494(19)-SGF29_P25554(32) 4   11 14 4.47E-12 KLNFLNMSK(1)-KEGGDNTPSK(1) inter ADA3_P32494(225)-GCN5_Q03330(283) 4   14 5 4.41E-11 TISKSHIVRPGLEQFK(4)-NPKSEFVVSQTLPR(3) inter ADA3_P32494(318)-SPT7_P35177(400)    3  2.56E-05 QSNLDLTVNLGIENLSLKHLLSSIQQK(18)-FLKMK(3) inter GCN5_Q03330(111)-ADA3_P32494(644)     3 1.39E-09 VVNNDNTKENMMVLTGLK(8)-AANSSLKSLLDK(7) 164  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence inter GCN5_Q03330(111)-SPT7_P35177(797)    6  3.31E-12 VVNNDNTKENMMVLTGLK(8)-QKIEQNSIMK(2) inter GCN5_Q03330(111)-SPT7_P35177(805) 2  1 15 12 6.09E-16 VVNNDNTKENMMVLTGLK(8)-IEQNSIMKNGFGTVLK(8) inter SGF11_Q03067(39)-SUS1_Q6WNK7(47)  2 1   2.81E-05 DLTKSEMNINESTNFTQILSTVEPK(4)-ETTQQQLLKTR(9) inter SGF11_Q03067(63)-UBP8_P50102(441) 2    1 7.36E-09 SYYFDPNGSLDINGLQKQQESSQYIHCENCGR(17)-ISGGQWFKFNDSMVSSISQEEVLK(8) inter SGF73_P53165(448)-SPT20_P50875(479)    7 7 7.46E-12 TPQPINHLTNQNLNPKQIQR(16)-LKFIEQWR(2) inter SGF73_P53165(545)-TRA1_P38811(2984)    3  5.60E-06 SQDTGLTPLEIQSQQQKLR(17)-IYTLPNIEIQEAFLKLR(15) inter SGF73_P53165(555)-TRA1_P38811(2984)    10 8 2.79E-09 QQQLQQQKFEAAASYLANATK(8)-IYTLPNIEIQEAFLKLR(15) inter SPT20_P50875(161)-SGF73_P53165(448)    5 5 1.14E-12 TPQPINHLTNQNLNPKQIQR(16)-LQQQQKQPELTSDGLILTK(6) inter SPT20_P50875(272)-TRA1_P38811(2713)     5 1.00E-10 AQQLYEVAQVKAR(11)-KASFK(1) inter SPT20_P50875(355)-TAF5_P38129(512)     9 1.05E-08 IWSLDGSSLNNPNIALNNNDKDEDPTCK(21)-KSFIHEHR(1) inter SPT20_P50875(91)-SGF73_P53165(401)    4 2 9.09E-16 KQQALQNYEAQFYQMLMTLNK(1)-EMFASSFSVKPGYTSPGYGAIHSR(10) inter SPT20_P50875(91)-SGF73_P53165(448)    1 3 4.10E-08 KQQALQNYEAQFYQMLMTLNK(1)-TPQPINHLTNQNLNPKQIQR(16) inter SPT3_P06844(1)-TRA1_P38811(2370) 2   5 5 1.36E-06 SWIFNTEIFPTVKEK(13)-MMDK(1) inter SPT3_P06844(301)-TRA1_P38811(2370) 5 5 6 27 30 1.79E-15 RLFDGPENVINPLKPR(14)-SWIFNTEIFPTVKEK(13) inter SPT7_P35177(409)-ADA3_P32494(663)    10 7 3.03E-10 IGPLFDKPEIMK(7)-HLLSSIQQKK(9) inter SPT7_P35177(409)-TAF5_P38129(754)    27 29 3.88E-16 ATTEPSAEPDEPFIGYLGDVTASINQDIKEYGR(29)-HLLSSIQQKK(9) inter SPT7_P35177(410)-TAF5_P38129(775)    10 3 8.54E-10 TVIPTSDLVASFYTKK(15)-KSQLGISDYELK(1) 165  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence inter SPT7_P35177(797)-TRA1_P38811(3175)    3 2 4.01E-07 TTKEDFAVIQR(3)-QKIEQNSIMK(2) inter SPT8_P38915(201)-SPT7_P35177(1240)    8 3 4.39E-06 KYDLLNTLEGK(1)-TATKAR(4) inter SPT8_P38915(244)-SPT7_P35177(1193)    10  8.04E-07 GMLDAGSFWNTLLPLLQKDYER(18)-KSEMK(1) inter SPT8_P38915(248)-SPT7_P35177(1255)    3 2 5.65E-08 KKPIASAFILPEEDLENDVK(1)-KSEMKLSANK(5) inter SUS1_Q6WNK7(2)-SGF73_P53165(9)    4 1 6.92E-08 SGDAEIKGIKPK(7)-TMDTAQLK(1) inter TAF10_Q12030(126)-SPT7_P35177(1069)    4 3 7.41E-09 FVSDIAKDAYEYSR(7)-KLQDIK(1) inter TAF10_Q12030(175)-SPT7_P35177(1128)     4 1.73E-14 ELGLEKEFGVLSSSVPLQLLTTQFQTVDGETK(6)-QLLQGQQQPGVQQISQQQHQQNEKTTASK(24) inter TAF10_Q12030(175)-TAF12_Q03761(526) 9 2 2 5 7 3.66E-16 QLLQGQQQPGVQQISQQQHQQNEKTTASK(24)-VAAAKNNGNNVASLNTK(5) inter TAF12_Q03761(131)-TRA1_P38811(2984)    5 1 5.91E-09 IYTLPNIEIQEAFLKLR(15)-NNSNKFSNMIK(5) inter TAF12_Q03761(284)-TRA1_P38811(2984) 1   5 2 1.21E-05 IYTLPNIEIQEAFLKLR(15)-KISSSNSTEIPSVTGPDALK(1) inter TAF12_Q03761(331)-TRA1_P38811(2878) 3   4  7.31E-09 GNVNTSQTEQSKAK(12)-AQEIKR(5) inter TAF12_Q03761(331)-TRA1_P38811(2984) 1   8 5 4.82E-10 IYTLPNIEIQEAFLKLR(15)-GNVNTSQTEQSKAK(12) inter TAF12_Q03761(333)-TRA1_P38811(2984)    7 8 1.48E-09 IYTLPNIEIQEAFLKLR(15)-AKVTNVNATASMLNNISSSK(2) inter TAF12_Q03761(351)-TRA1_P38811(2878)    3 2 5.36E-06 VTNVNATASMLNNISSSKSAIFK(18)-AQEIKR(5) inter TAF12_Q03761(351)-TRA1_P38811(832)    4  2.61E-07 VTNVNATASMLNNISSSKSAIFK(18)-SIKPILQVLLQSLNQMILTAR(3) inter TAF12_Q03761(371)-TRA1_P38811(2781)     3 2.64E-08 VADWNSDRDALEQSVKSVMDVPTPR(16)-QTEPAIPISENISTKTPAPVAYR(15) inter TAF12_Q03761(371)-TRA1_P38811(2795) 3 2  4 17 6.97E-15 QTEPAIPISENISTKTPAPVAYR(15)-RQMFKTFLALQNFAESR(5) 166  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence inter TAF12_Q03761(371)-TRA1_P38811(2815) 2 3 5 12 10 3.22E-14 QTEPAIPISENISTKTPAPVAYR(15)-KLCDEGIQLSLIK(1) inter TAF12_Q03761(403)-SPT20_P50875(415) 2 1   2 1.84E-16 SNRPTITGGSAMNASALNTPATTKLPPYEMDTQR(24)-TTTITNSTFAVSLTKNAMEIASSSSNGVR(15) inter TAF12_Q03761(403)-TRA1_P38811(2589)    4 2 1.03E-05 SNRPTITGGSAMNASALNTPATTKLPPYEMDTQR(24)-SIITLLSKPYHTR(8) inter TAF5_P38129(512)-SPT7_P35177(889) 2  4 2 1 3.39E-05 IWSLDGSSLNNPNIALNNNDKDEDPTCK(21)-MLQNGINKQSR(8) inter TAF5_P38129(70)-SGF73_P53165(401) 11 10 11 23 16 7.59E-10 EMFASSFSVKPGYTSPGYGAIHSR(10)-IVLEYLNKK(8) inter TAF5_P38129(70)-SGF73_P53165(448) 2   6 11 1.04E-12 TPQPINHLTNQNLNPKQIQR(16)-IVLEYLNKK(8) inter TAF5_P38129(70)-SPT20_P50875(161) 1   5 2 4.13E-06 LQQQQKQPELTSDGLILTK(6)-IVLEYLNKK(8) inter TAF5_P38129(70)-SPT20_P50875(91)    15 14 1.16E-13 KQQALQNYEAQFYQMLMTLNK(1)-IVLEYLNKK(8) inter TAF5_P38129(775)-SPT7_P35177(869)    3 8 7.01E-13 FLQEYDISNAIPDIVYEGVNTKTLDK(22)-RTVIPTSDLVASFYTKK(16) inter TAF5_P38129(776)-SPT7_P35177(869) 2  1 14 1 3.26E-10 FLQEYDISNAIPDIVYEGVNTKTLDK(22)-KTPVFK(1) inter TAF5_P38129(93)-SPT20_P50875(111)    3 5 1.01E-08 QQALQNYEAQFYQMLMTLNKRPK(20)-TLTPQNKQSPANTK(7) inter TAF6_P53040(221)-SPT7_P35177(760) 7 8 7 20 17 1.28E-14 HVLSKELQIYFNK(5)-NGKLNSDSEAFLK(3) inter TAF6_P53040(229)-SPT7_P35177(760)  9 8 24 31 9.72E-15 ELQIYFNKVISTLTAK(8)-NGKLNSDSEAFLK(3) inter TAF6_P53040(229)-TAF5_P38129(437)     4 3.58E-05 ELQIYFNKVISTLTAK(8)-LEIQKVK(5) inter TAF6_P53040(237)-SPT7_P35177(760)    2 3 2.41E-12 VISTLTAKSQADEAAQHMK(8)-NGKLNSDSEAFLK(3) inter TAF6_P53040(506)-TRA1_P38811(2193)    4  3.84E-07 EWIMENLPTIQNLLEKCIK(16)-RDDAKELISAIFFGE(5) inter TAF9_Q05027(106)-SPT20_P50875(479)    2  1.55E-04 TQYQFKPTAPK(6)-LKFIEQWR(2) 167  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence inter TAF9_Q05027(106)-TAF5_P38129(547)  1 2 7 3 3.06E-07 YLLSGSEDKTVR(9)-TQYQFKPTAPK(6) inter TAF9_Q05027(11)-ADA1_Q12060(183)    5 7 1.08E-19 NVLNKNSVGSVSEVGPDSTQEETPR(5)-KIVMSLPLNDR(1) inter TAF9_Q05027(155)-SPT20_P50875(355)    2 2 3.15E-06 EWDLEDPKSM(8)-KSFIHEHR(1) inter TAF9_Q05027(69)-SPT3_P06844(333)     4 5.74E-06 YTQGVLKDALVYNDYAGSGNSAGSGLGVEDIR(7)-LSSKPIIM(4) inter UBP8_P50102(15)-SGF73_P53165(33)    2  5.96E-07 VIEEYSLSQGSGPSNDSWKSLMSSAK(19)-SICPHIQQVFQNEKSK(14) inter UBP8_P50102(2)-SGF11_Q03067(39)    7 4 2.33E-08 SICPHIQQVFQNEK(1)-ETTQQQLLKTR(9) inter UBP8_P50102(22)-SGF73_P53165(33) 11 7 3 25 27 1.28E-13 VIEEYSLSQGSGPSNDSWKSLMSSAK(19)-DGVLKTCNAAR(5) inter UBP8_P50102(22)-SGF73_P53165(40) 10 2  30 19 1.11E-13 SLMSSAKDTPLQYDHMNR(7)-DGVLKTCNAAR(5) intra ADA2_Q02336(238)-ADA2_Q02336(242) 4   4  4.02E-05 LQAIDKK(6)-SKEAK(2) intra SPT8_P38915(503)-SPT8_P38915(548) 1    1 2.64E-05 MPSKPIHNLKLPSISGPVSCVK(10)-LYNVEIAVDASNSTTKSSK(16) intra ADA1_Q12060(234)-ADA1_Q12060(237) 7 9 9 14 10 5.04E-12 IPIVTNPESLKR(11)-VKSNNLK(2) intra ADA2_Q02336(232)-ADA2_Q02336(268)    6 6 3.71E-08 VMTAQDFEEFSKDILEELHCR(12)-KLQAIDK(1) intra ADA2_Q02336(238)-ADA2_Q02336(245) 1 2  6  2.84E-05 EAKELYNR(3)-LQAIDKK(6) intra ADA2_Q02336(239)-ADA2_Q02336(242)    2  2.60E-06 LQAIDKKR(7)-SKEAK(2) intra ADA3_P32494(19)-ADA3_P32494(35) 1 5 2 5 12 3.96E-12 LLSSMLKTLDLTFER(7)-KEGGDNTPSK(1) intra ADA3_P32494(206)-ADA3_P32494(209)  3 3   8.44E-07 ATENENTQRPDNKK(13)-QKIDVDK(2) intra ADA3_P32494(214)-ADA3_P32494(225)    2  1.12E-04 QKIDVDKMENDPTVK(7)-NPKSEFVVSQTLPR(3) 168  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra ADA3_P32494(258)-ADA3_P32494(260) 7 4 4 10 6 9.42E-12 AAAALGLFNEEGLESTGEDFLKK(22)-KYNVASYPTNDLK(1) intra ADA3_P32494(315)-ADA3_P32494(320) 4 4 3 18 15 1.02E-08 MKYIIPDSLQFDK(2)-DLSDDNLKFLK(8) intra ADA3_P32494(315)-ADA3_P32494(331)  1  7 3 1.50E-05 YIIPDSLQFDKTYDPEVNPFIIPK(11)-DLSDDNLKFLK(8) intra ADA3_P32494(320)-ADA3_P32494(563)    2  4.69E-05 MKYIIPDSLQFDK(2)-ELQGTLKQVTK(7) intra ADA3_P32494(355)-ADA3_P32494(365)     5 2.67E-19 NSAYKKPSPYSNDASTILPK(5)-LGPLYTDVWFKDENDK(11) intra ADA3_P32494(360)-ADA3_P32494(366) 3 2  8 1 3.78E-11 LGPLYTDVWFKDENDKNSAYK(16)-KPSPYSNDASTILPK(1) intra ADA3_P32494(366)-ADA3_P32494(381)    8 6 5.23E-15 KSANELDDNALESGSISCGPLLSR(1)-KPSPYSNDASTILPK(1) intra ADA3_P32494(92)-ADA3_P32494(100)    2 2 2.24E-07 NEKQANDEK(3)-KIRDSK(1) intra GCN5_Q03330(111)-GCN5_Q03330(153) 3 7 9 26 23 1.20E-17 VVNNDNTKENMMVLTGLK(8)-KPLTVVGGITYRPFDK(1) intra GCN5_Q03330(223)-GCN5_Q03330(328)    9 5 1.49E-12 EAGWTPEMDALAQRPKR(16)-KQGFTK(1) intra GCN5_Q03330(223)-GCN5_Q03330(385) 5 4 3 5 3 1.05E-07 LESNKYQK(5)-KQGFTK(1) intra GCN5_Q03330(223)-GCN5_Q03330(388)    4 2 7.51E-09 YQKMEDFIYDAR(3)-KQGFTK(1) intra GCN5_Q03330(228)-GCN5_Q03330(385)  2   3 1.01E-04 QGFTKEITLDK(5)-LESNKYQK(5) intra GCN5_Q03330(228)-GCN5_Q03330(429)     2 1.03E-04 VKEIPEYSHLID(2)-QGFTKEITLDK(5) intra GCN5_Q03330(328)-GCN5_Q03330(385)  4 3 4 4 1.52E-08 EAGWTPEMDALAQRPKR(16)-LESNKYQK(5) intra GCN5_Q03330(415)-GCN5_Q03330(422) 10 10 12 24 25 1.38E-12 MYNGENTSYYKYANR(11)-LEKFFNNK(3) intra GCN5_Q03330(422)-GCN5_Q03330(429) 10 13 11 30 31 7.45E-09 VKEIPEYSHLID(2)-LEKFFNNK(3) 169  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra GCN5_Q03330(48)-GCN5_Q03330(53)  1  2  5.76E-08 LENNVEEIQPEQAETNKQEGTDKENK(23)-GKFEK(2) intra GCN5_Q03330(72)-GCN5_Q03330(87) 5  7   2.31E-08 FEFDGVEYTFKERPSVVEENEGK(11)-IGGSEVVTDVEKGIVK(12) intra GCN5_Q03330(72)-GCN5_Q03330(99)     2 1.57E-09 ERPSVVEENEGKIEFR(12)-IGGSEVVTDVEKGIVK(12) intra SGF29_P25554(104)-SGF29_P25554(109) 3 6 4 6  1.26E-14 MALSQGKK(7)-AVGKVGR(4) intra SGF29_P25554(109)-SGF29_P25554(181) 2  3 5 4 4.17E-06 KELLLIPPGFPTK(1)-AVGKVGR(4) intra SGF29_P25554(134)-SGF29_P25554(181) 9 10 10 15 19 4.23E-20 SYWTSEYNPNAPILVGSEVAYKPR(22)-KELLLIPPGFPTK(1) intra SGF29_P25554(151)-SGF29_P25554(177)  1 2  4 3.60E-07 RGSADGEWIQCEVLKVVADGTR(15)-VYKCNR(3) intra SGF29_P25554(181)-SGF29_P25554(220)    1 1 1.20E-08 YPETTTFYPAIVIGTKR(16)-KELLLIPPGFPTK(1) intra SGF73_P53165(231)-SGF73_P53165(265)    18 14 6.05E-23 HLIDFNKQCGVELPEGGYCAR(7)-SKPYDVLLADYHR(2) intra SGF73_P53165(250)-SGF73_P53165(281)    2  1.98E-07 SLTCKSHSMGAK(5)-EHQTKIGAAAEK(5) intra SGF73_P53165(288)-SGF73_P53165(291) 1  3 1  1.18E-08 AKQQELQK(2)-IGAAAEKR(7) intra SGF73_P53165(304)-SGF73_P53165(308)    1 1 2.56E-04 QIQKEQK(4)-KHTQQQK(1) intra SGF73_P53165(304)-SGF73_P53165(314)    4 3 4.63E-05 HTQQQKQGQR(6)-QIQKEQKK(4) intra SGF73_P53165(428)-SGF73_P53165(448) 1   10  9.69E-08 TPQPINHLTNQNLNPKQIQR(16)-TTDYKFR(5) intra SGF73_P53165(545)-SGF73_P53165(555) 2   8 4 1.37E-17 QQQLQQQKFEAAASYLANATK(8)-SQDTGLTPLEIQSQQQKLR(17) intra SPT20_P50875(161)-SPT20_P50875(230)    3 4 1.69E-17 ENKPNLNSSSSPSNNNSTQDNSK(3)-LQQQQKQPELTSDGLILTK(6) intra SPT20_P50875(161)-SPT20_P50875(271)    3  1.52E-04 IQQPSEPNSGVANTGANTANKK(21)-LQQQQKQPELTSDGLILTK(6) 170  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra SPT20_P50875(161)-SPT20_P50875(272)  4  12 10 1.50E-06 LQQQQKQPELTSDGLILTK(6)-KASFK(1) intra SPT20_P50875(161)-SPT20_P50875(479)    7 5 4.92E-11 LQQQQKQPELTSDGLILTK(6)-LKFIEQWR(2) intra SPT20_P50875(230)-SPT20_P50875(272) 1 3 7 6 4 7.86E-07 ENKPNLNSSSSPSNNNSTQDNSK(3)-KASFK(1) intra SPT20_P50875(271)-SPT20_P50875(276) 4 4  3  1.09E-05 IQQPSEPNSGVANTGANTANKK(21)-ASFKRPR(4) intra SPT20_P50875(91)-SPT20_P50875(161)    4 6 1.16E-18 KQQALQNYEAQFYQMLMTLNK(1)-LQQQQKQPELTSDGLILTK(6) intra SPT3_P06844(1)-SPT3_P06844(6)   3 5 8 2.48E-05 HKYR(2)-MMDK(1) intra SPT3_P06844(116)-SPT3_P06844(123)  1 2   6.31E-07 DQDASAGVASGTGNPGAGGEDDLKK(24)-AGGGEKDEK(6) intra SPT3_P06844(133)-SPT3_P06844(136)    3 8 2.13E-08 DGGNMMKVK(7)-KSQIK(1) intra SPT3_P06844(176)-SPT3_P06844(179) 3 3 3 4  1.29E-11 EANIVTLKR(8)-LKMADDR(2) intra SPT3_P06844(85)-SPT3_P06844(92)    3 3 1.24E-08 NAKDQDASAGVASGTGNPGAGGEDDLK(3)-TYLSWKDLR(6) intra SPT7_P35177(1065)-SPT7_P35177(1069)    9 7 2.74E-09 GTNVEMLQTTLLENGINRPDDLFSYVESEFGKK(32)-KLQDIK(1) intra SPT7_P35177(1069)-SPT7_P35177(1076) 9 9 8 15 19 6.39E-11 QKLESFLR(2)-KLQDIK(1) intra SPT7_P35177(1076)-SPT7_P35177(1154)    4 3 1.12E-07 EFGVLSSSVPLQLLTTQFQTVDGETKVQAK(26)-QKLESFLR(2) intra SPT7_P35177(1175)-SPT7_P35177(1199)    1 4 2.86E-08 ITKGMLDAGSFWNTLLPLLQK(3)-SKAYIAK(2) intra SPT7_P35177(1193)-SPT7_P35177(1204) 1  3 14 6 9.54E-09 GMLDAGSFWNTLLPLLQKDYER(18)-AYIAKQSK(5) intra SPT7_P35177(1193)-SPT7_P35177(1236)    10 8 5.07E-08 GMLDAGSFWNTLLPLLQKDYER(18)-KTATK(1) intra SPT7_P35177(1199)-SPT7_P35177(1207) 8 5 4 8  6.83E-11 QSKSSANDK(3)-SKAYIAK(2) 171  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra SPT7_P35177(1199)-SPT7_P35177(1213)     2 1.70E-07 SSANDKTSMTSTEDNSFALLEEDQFVSK(6)-SKAYIAK(2) intra SPT7_P35177(1204)-SPT7_P35177(1235) 10 9 10 12 19 2.30E-10 TSMTSTEDNSFALLEEDQFVSKK(22)-AYIAKQSK(5) intra SPT7_P35177(1204)-SPT7_P35177(1236) 5  4 8 5 1.40E-13 AYIAKQSK(5)-KTATK(1) intra SPT7_P35177(1207)-SPT7_P35177(1236) 3 3  2 15 2.05E-11 QSKSSANDK(3)-KTATK(1) intra SPT7_P35177(1235)-SPT7_P35177(1254)     4 3.02E-04 TSMTSTEDNSFALLEEDQFVSKK(22)-ISTTYKK(6) intra SPT7_P35177(1236)-SPT7_P35177(1254)   3 4 3 4.96E-07 ISTTYKK(6)-KTATK(1) intra SPT7_P35177(1240)-SPT7_P35177(1254) 1 1   1 1.58E-07 ISTTYKK(6)-TATKAR(4) intra SPT7_P35177(16)-SPT7_P35177(24)  3 5 12 8 2.78E-09 LTEKLFNK(4)-TNAKALLK(4) intra SPT7_P35177(222)-SPT7_P35177(231)  3    2.36E-05 EGISKFAEDEDYDDEDENYDEDSTDVK(5)-EEEEIGKNEKPQNK(10) intra SPT7_P35177(286)-SPT7_P35177(293)   5   1.59E-07 LKTNNVEEIMGNWNK(2)-LVLNISISKETLSK(9) intra SPT7_P35177(293)-SPT7_P35177(538)    14 8 1.34E-10 LKTNNVEEIMGNWNK(2)-KSLQLIR(1) intra SPT7_P35177(315)-SPT7_P35177(323) 6    10 3.18E-07 IYHSFEYDKETMIK(9)-RLKLEESDK(3) intra SPT7_P35177(410)-SPT7_P35177(797)    2  6.82E-06 KSQLGISDYELK(1)-QKIEQNSIMK(2) intra SPT7_P35177(421)-SPT7_P35177(789)    1 2 6.80E-07 SQLGISDYELKHLIMDVR(11)-EQKALESYR(3) intra SPT7_P35177(433)-SPT7_P35177(493)    3  1.14E-07 SMDLNTVLKK(9)-SKWTSDER(2) intra SPT7_P35177(433)-SPT7_P35177(496) 3 5 9 7 3 1.68E-10 LKSFQYDSK(2)-SKWTSDER(2) intra SPT7_P35177(451)-SPT7_P35177(538) 1 8 9  4 4.79E-09 IGQEELYEACEKVVLELR(12)-KSLQLIR(1) 172  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra SPT7_P35177(469)-SPT7_P35177(493) 4   5 2 2.42E-09 NYTEHSTPFLNKVSK(12)-SMDLNTVLKK(9) intra SPT7_P35177(493)-SPT7_P35177(496) 4 10 9 14 12 2.16E-18 SMDLNTVLKK(9)-LKSFQYDSK(2) intra SPT7_P35177(538)-SPT7_P35177(836)    2  5.07E-08 QEDDDQLQFHNDHSLNGNEAFEKQPNDIELDDTR(23)-KSLQLIR(1) intra SPT7_P35177(603)-SPT7_P35177(610)    3  4.46E-06 TVKDEAPTNDDK(3)-VSEKDSSK(4) intra SPT7_P35177(673)-SPT7_P35177(680) 5 10 10 4  1.42E-12 TEDSSKDADAAK(6)-KDTEDGLQDK(1) intra SPT7_P35177(673)-SPT7_P35177(689)  2 1   1.15E-06 DTEDGLQDKTAENK(9)-TEDSSKDADAAKK(6) intra SPT7_P35177(679)-SPT7_P35177(689)   1 4  9.47E-05 DTEDGLQDKTAENK(9)-DADAAKK(6) intra SPT7_P35177(743)-SPT7_P35177(751) 4 4 4 5 1 7.82E-09 AEICLKR(6)-TVTAKVR(5) intra SPT7_P35177(797)-SPT7_P35177(836) 3     4.67E-06 QEDDDQLQFHNDHSLNGNEAFEKQPNDIELDDTR(23)-QKIEQNSIMK(2) intra SPT7_P35177(8)-SPT7_P35177(16)  4 3 4 4 2.97E-08 IPIKNYQR(4)-TNAKALLK(4) intra SPT7_P35177(8)-SPT7_P35177(24) 1 2 2 2 4 3.98E-06 IPIKNYQR(4)-LTEKLFNK(4) intra SPT7_P35177(8)-SPT7_P35177(57)  4 9  4 4.08E-08 LKAWDYFLK(2)-IPIKNYQR(4) intra SPT7_P35177(889)-SPT7_P35177(897)  3 2   1.01E-06 FLANKDLGLTPK(5)-MLQNGINKQSR(8) intra SPT7_P35177(921)-SPT7_P35177(1074) 3 3 8 2 17 4.14E-07 HICHKISLIR(5)-KLQDIKQK(6) intra SPT7_P35177(921)-SPT7_P35177(1128)     4 1.34E-06 ELGLEKEFGVLSSSVPLQLLTTQFQTVDGETK(6)-HICHKISLIR(5) intra SPT8_P38915(188)-SPT8_P38915(201)   5 1  7.08E-14 GLKYLFLGGSDGYIR(3)-KYDLLNTLEGK(1) intra SPT8_P38915(244)-SPT8_P38915(253) 6 5 8 16 12 1.08E-08 LSANKTDYEPK(5)-KSEMK(1) 173  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra SPT8_P38915(329)-SPT8_P38915(346) 5 3 3 11 10 7.85E-08 LLEWDLQTGDIVNEFKK(16)-FLSGSWDKR(8) intra SPT8_P38915(521)-SPT8_P38915(551)     4 4.38E-11 SSKVPFLIVPGHHGGIISNLYLDPTSR(3)-AMPNNKHLLCASR(6) intra SUS1_Q6WNK7(30)-SUS1_Q6WNK7(43)  8 10 3 15 1.43E-10 SQIQQYLVESGNYELISNELKAR(21)-VKDLTK(2) intra SUS1_Q6WNK7(41)-SUS1_Q6WNK7(68)    1 10 1.49E-09 SEMNINESTNFTQILSTVEPKALEMVSDSTR(21)-LLQEGWVDKVK(9) intra TAF12_Q03761(131)-TAF12_Q03761(221) 1   16 12 1.58E-19 NNSNKFSNMIK(5)-LFNNYQNSKK(9) intra TAF12_Q03761(131)-TAF12_Q03761(222)    3  1.18E-05 NNSNKFSNMIK(5)-KTFYVECAR(1) intra TAF12_Q03761(137)-TAF12_Q03761(221) 1   16 15 2.12E-09 FSNMIKQVLTPEENQEYEK(6)-LFNNYQNSKK(9) intra TAF12_Q03761(163)-TAF12_Q03761(170) 1    5 5.23E-06 ETYLKQNIDRLEQEINK(5)-HTSIKEK(5) intra TAF12_Q03761(165)-TAF12_Q03761(195)  1  4 5 1.37E-05 EKETYLK(2)-QQLQEKK(6) intra TAF12_Q03761(165)-TAF12_Q03761(212)    2  1.87E-07 IEYTKLFNNYQNSK(5)-EKETYLK(2) intra TAF12_Q03761(170)-TAF12_Q03761(196)    17 15 3.97E-13 ETYLKQNIDRLEQEINK(5)-KIELLNDWK(1) intra TAF12_Q03761(170)-TAF12_Q03761(207)    1 3 5.86E-05 ETYLKQNIDRLEQEINK(5)-VLKIEYTK(3) intra TAF12_Q03761(182)-TAF12_Q03761(196)    1 4 1.21E-08 QNIDRLEQEINKQTDEGPKQQLQEK(12)-KIELLNDWK(1) intra TAF12_Q03761(189)-TAF12_Q03761(196) 8 3 2 19 22 9.71E-13 QNIDRLEQEINKQTDEGPKQQLQEK(19)-KIELLNDWK(1) intra TAF12_Q03761(196)-TAF12_Q03761(207)    2  1.10E-04 KIELLNDWK(1)-VLKIEYTK(3) intra TAF12_Q03761(44)-TAF12_Q03761(71) 6 7 4 20 15 4.01E-10 QLQEMAAKFR(8)-ELMFQAAKIK(8) intra TAF12_Q03761(466)-TAF12_Q03761(469)   2   1.20E-04 KSDNLEAR(1)-LAKHR(3) 174  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra TAF5_P38129(103)-TAF5_P38129(119) 15   16 10 2.14E-17 TGKFPEQSSIPPNPGK(3)-TAKPISNPTNLSSK(3) intra TAF5_P38129(103)-TAF5_P38129(130)    2  1.80E-05 TGKFPEQSSIPPNPGK(3)-TAKPISNPTNLSSKR(14) intra TAF5_P38129(130)-TAF5_P38129(432) 2   3 3 7.36E-06 TAKPISNPTNLSSKR(14)-TALDLKLEIQK(6) intra TAF5_P38129(209)-TAF5_P38129(290)    13 6 5.26E-07 FSPDFKDFHGSEINR(6)-EKLADGIK(2) intra TAF5_P38129(209)-TAF5_P38129(307)    4  1.60E-09 VLSDSENGNGKQNLEMNSVPVK(11)-FSPDFKDFHGSEINR(6) intra TAF5_P38129(229)-TAF5_P38129(296)     12 1.14E-11 LFSVNSIDHIKENEVASAFQSHK(11)-LADGIKVLSDSENGNGK(6) intra TAF5_P38129(241)-TAF5_P38129(432) 8 2 4 10 10 4.14E-12 ENEVASAFQSHKYR(12)-TALDLKLEIQK(6) intra TAF5_P38129(290)-TAF5_P38129(307)    8 2 3.35E-07 VLSDSENGNGKQNLEMNSVPVK(11)-EKLADGIK(2) intra TAF5_P38129(318)-TAF5_P38129(330)    9 8 4.85E-12 QNLEMNSVPVKLGPFPK(11)-DEEFVKEIETELK(6) intra TAF5_P38129(324)-TAF5_P38129(368)    4  1.48E-06 TLLQEYKAMNNEK(7)-LGPFPKDEEFVK(6) intra TAF5_P38129(330)-TAF5_P38129(339)    3 12 1.90E-08 DEEFVKEIETELK(6)-IKDDQEK(2) intra TAF5_P38129(339)-TAF5_P38129(368)    2  6.71E-08 TLLQEYKAMNNEK(7)-IKDDQEK(2) intra TAF5_P38129(339)-TAF5_P38129(432)  6 7 3 5 5.94E-08 TALDLKLEIQK(6)-IKDDQEK(2) intra TAF5_P38129(344)-TAF5_P38129(368)    6 4 2.19E-12 IKDDQEKQLNQQTAGDNYSGANNR(7)-TLLQEYKAMNNEK(7) intra TAF5_P38129(368)-TAF5_P38129(376) 4 5 7 18 17 4.37E-13 TLLQEYKAMNNEK(7)-FKDNTGDDDK(2) intra TAF5_P38129(393)-TAF5_P38129(398)   4 8 1 4.59E-06 IAKDEEK(3)-KESELK(1) intra TAF5_P38129(403)-TAF5_P38129(409)    3  1.25E-08 KESELKVDGEK(6)-KDSNLSSPAR(1) 175  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra TAF5_P38129(426)-TAF5_P38129(437)    14 12 3.83E-07 DILPLPPKTALDLK(8)-LEIQKVK(5) intra TAF5_P38129(432)-TAF5_P38129(439) 2 6 6 3 8 4.94E-05 TALDLKLEIQK(6)-VKESR(2) intra TAF5_P38129(93)-TAF5_P38129(130) 6 1 2  6 6.14E-07 TAKPISNPTNLSSKR(14)-TLTPQNKQSPANTK(7) intra TAF5_P38129(93)-TAF5_P38129(446) 1 1 2 8 6 4.51E-13 DAIKLDNLQLALPSVCMYTFQNTNK(4)-TLTPQNKQSPANTK(7) intra TAF6_P53040(237)-TAF6_P53040(329)  3 3 10 25 1.06E-13 KLGGSPKDDSPQEIHEFLER(7)-VISTLTAKSQADEAAQHMK(8) intra TAF6_P53040(359)-TAF6_P53040(369)     4 3.79E-08 DFAASLLDYVLKK(12)-SLKPR(3) intra TAF6_P53040(359)-TAF6_P53040(466)    6 11 7.56E-17 DFAASLLDYVLKK(12)-KDLPDLYEGK(1) intra TAF6_P53040(484)-TAF6_P53040(501)   3  2 1.37E-05 CGVTIGFHILKR(11)-VTDEDKEK(6) intra TRA1_P38811(1051)-TRA1_P38811(1232)    9 12 1.30E-07 KYQYNLANGLLFVLK(1)-TAVNSIKLER(7) intra TRA1_P38811(1059)-TRA1_P38811(3295)    3 16 3.73E-16 FSTTLLAPYIRPKFNADFIDNKPDYETYIK(13)-IGIEKNFDLEPTVNKR(5) intra TRA1_P38811(1059)-TRA1_P38811(3304)    8  1.61E-05 FNADFIDNKPDYETYIKR(9)-IGIEKNFDLEPTVNKR(5) intra TRA1_P38811(1059)-TRA1_P38811(3312)   1 8 2 2.39E-11 FNADFIDNKPDYETYIKR(17)-IGIEKNFDLEPTVNKR(5) intra TRA1_P38811(1169)-TRA1_P38811(1174)   1 5 3 7.52E-06 IYEKSCLIYGEELALSHSFIPELAK(4)-EVGVLAYKR(8) intra TRA1_P38811(1169)-TRA1_P38811(1224) 4 4 3 5 8 3.50E-12 VLIDNVKSSSVFLK(7)-EVGVLAYKR(8) intra TRA1_P38811(1340)-TRA1_P38811(3348)    1 9 1.22E-05 ENLEVLCPHLSNFHHQKFEDIEIPGQYLLNK(17)-QFLLSPIFAKPLR(10) intra TRA1_P38811(1483)-TRA1_P38811(1521)    17 8 1.22E-17 ELLQNGLKPLLMNLSDHQK(8)-KLLDHLTAWCR(1) intra TRA1_P38811(1613)-TRA1_P38811(1657) 5 4 2 12 16 9.20E-12 ELDNFYDFYISNIPKNQVR(15)-KNMTLR(1) 176  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra TRA1_P38811(2569)-TRA1_P38811(2576)    13 1 2.31E-09 AWVTLFPQVYKSIPK(11)-NEKYGFVR(3) intra TRA1_P38811(2781)-TRA1_P38811(2808)     7 7.39E-13 VADWNSDRDALEQSVKSVMDVPTPR(16)-KGDQEVR(1) intra TRA1_P38811(2781)-TRA1_P38811(2815)     2 4.54E-12 VADWNSDRDALEQSVKSVMDVPTPR(16)-KLCDEGIQLSLIK(1) intra TRA1_P38811(2795)-TRA1_P38811(2808) 5    3 8.92E-10 RQMFKTFLALQNFAESR(5)-KGDQEVR(1) intra TRA1_P38811(2795)-TRA1_P38811(2815)    7 2 1.59E-13 RQMFKTFLALQNFAESR(5)-KLCDEGIQLSLIK(1) intra TRA1_P38811(2808)-TRA1_P38811(2815) 1 4 1 12 6 9.89E-13 KLCDEGIQLSLIK(1)-KGDQEVR(1) intra TRA1_P38811(2956)-TRA1_P38811(3432)    5  1.97E-07 KHNMPDVCISQLAR(1)-SLSKNVETR(4) intra TRA1_P38811(3175)-TRA1_P38811(3192) 8 5  13 12 8.46E-14 QTMAVMGDKPDTNDR(9)-TTKEDFAVIQR(3) intra TRA1_P38811(3175)-TRA1_P38811(3238)     3 5.89E-09 FKSTTDEDLFR(2)-TTKEDFAVIQR(3) intra TRA1_P38811(3271)-TRA1_P38811(3304)    2  1.44E-05 FNADFIDNKPDYETYIK(9)-KNPKLPENTEK(4) intra TRA1_P38811(3278)-TRA1_P38811(3304)     3 9.52E-06 FNADFIDNKPDYETYIK(9)-LPENTEKNLVK(7) intra TRA1_P38811(3282)-TRA1_P38811(3304)    5 8 1.02E-21 FNADFIDNKPDYETYIK(9)-NLVKFSTTLLAPYIRPK(4) intra TRA1_P38811(3295)-TRA1_P38811(3324)     2 7.04E-05 FSTTLLAPYIRPKFNADFIDNKPDYETYIK(13)-RLENKLDR(5) intra TRA1_P38811(451)-TRA1_P38811(602) 3   17 9 4.64E-17 TIIHDLKVFNPPPNEYTVANPK(7)-LGKENPQEAPR(3) intra TRA1_P38811(520)-TRA1_P38811(2218) 10 3 6 1 2 7.44E-20 AIKAQGVSVIIEEESPGK(3)-KVLEPSDDDHLMPQPK(1) intra TRA1_P38811(536)-TRA1_P38811(1015)    2  3.42E-06 KEDINDSPDVEMTESDK(1)-KSAYK(1) intra TRA1_P38811(749)-TRA1_P38811(1302)    7 4 1.95E-13 VLENTLTDIVCELSNANPKVR(19)-LKDLGNVDFNTSNVLIR(2) 177  Table A.1. Results from crosslinking mass-spectrometry of SAGA   Sample 1  (Spectral count) Sample 2  (Spectral count)   Inter- or intra-molecular Cross-linked sites 80µM DSS 160µM DSS 320µM DSS 160µM DSS 320µM DSS Best E-value Peptide sequence intra TRA1_P38811(961)-TRA1_P38811(3362) 1   8 10 2.90E-09 FEDIEIPGQYLLNKDNNVHFIK(14)-QFLKPPTDLTEK(4) intra UBP8_P50102(15)-UBP8_P50102(22)    4  1.48E-08 SICPHIQQVFQNEKSK(14)-DGVLKTCNAAR(5) intra UBP8_P50102(22)-UBP8_P50102(116)    7 5 7.42E-10 CEDYIGNIDLINDAILAKYWDDVCTK(18)-DGVLKTCNAAR(5) intra UBP8_P50102(315)-UBP8_P50102(389)    4 4 7.92E-16 KLDDFIEFPTYLNMKNYCSTK(15)-KLYECLDSFHK(1) intra UBP8_P50102(326)-UBP8_P50102(363)    4 3 2.42E-10 LPSVLVLQLKR(10)-KEQLK(1) intra UBP8_P50102(326)-UBP8_P50102(395)    2  6.47E-07 NYCSTKEK(6)-KEQLK(1) intra UBP8_P50102(326)-UBP8_P50102(399)  2  3 13 1.88E-07 DKHSENGKVPDIIYELIGIVSHK(2)-KEQLK(1) intra UBP8_P50102(347)-UBP8_P50102(363)    3  2.85E-07 DFNYHCGECNSTQDAIKQLGIHK(17)-LPSVLVLQLKR(10)           178  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence inter EAF5_P39995(199)-TRA1_P38811(2713) 4 5  1 1.40E-12 AQQLYEVAQVKAR(11)-LESKTGNFQK(4):0 inter EPL1_P43572(810)-TRA1_P38811(2984)  5 5 8 1.22E-18 IYTLPNIEIQEAFLKLR(15)-KLNDSLINSEAK(1):0 inter SWC4_P53201(345)-TRA1_P38811(2589)    2 2.04E-05 SIITLLSKPYHTR(8)-QLQQLNVKK(8):0 inter EAF5_P39995(183)-TRA1_P38811(2984) 7 6 6 4 1.45E-06 IYTLPNIEIQEAFLKLR(15)-EKLLK(2):0 inter EAF1_Q06337(713)-TRA1_P38811(2370) 9 9 10 9 1.67E-08 SWIFNTEIFPTVKEK(13)-FNFSDLKGPR(7):0 inter YNG2_P38806(70)-EPL1_P43572(374)   2  9.00E-04 HPQEDGLDKEIK(9)-VKIK(2):0 inter YNG2_P38806(41)-EPL1_P43572(395)  1 2  9.79E-09 SLNISGEDDDLINHKR(15)-KKYEQK(2):0 inter ARP4_P80428(210)-EPL1_P43572(395)  1 1  5.62E-06 SLNISGEDDDLINHKR(15)-EIIPLFAIKQR(9):0 inter EAF1_Q06337(118)-TRA1_P38811(3175)    2 1.06E-08 TTKEDFAVIQR(3)-LSDSKMSR(5):0 inter SWC4_P53201(372)-EPL1_P43572(427)    4 3.97E-15 KSESAYAEQLLK(1)-AAAAAAAAKAK(9):0 inter SWC4_P53201(66)-EAF1_Q06337(425)   2 1 1.29E-05 ISEKSGRPSDTSR(4)-SGNNFKEK(6):0 inter EPL1_P43572(821)-TRA1_P38811(2984)   3  3.85E-11 LNDSLINSEAKQNSSITQK(11)-IYTLPNIEIQEAFLKLR(15):0 inter EAF1_Q06337(25)-EAF5_P39995(142) 1  4 1 7.92E-06 NGIKTGASQLQPHANAGK(4)-SKFPK(2):0 inter SWC4_P53201(66)-EAF1_Q06337(406)   3 8 9.81E-11 SGNNFKEK(6)-KTLLPGK(1):0 inter EPL1_P43572(569)-TRA1_P38811(3492)  7 4  9.49E-14 KGFDPDDIQDFMADKLNAAHDDALPAPDMTILK(15)-NFSTSNVPFASIASSKFQIDR(16):0 inter EPL1_P43572(569)-TRA1_P38811(3477)  4 5 10 2.07E-21 NFSTSNVPFASIASSKFQIDR(16)-KKGFDPDDIQDFMADK(1):0 inter EAF1_Q06337(291)-EPL1_P43572(748)   5 1 4.32E-10 IPQALPLAELKYMSQTLPLINLIPR(11)-FGTMLGTKSYEQLR(8):0 inter EAF1_Q06337(784)-TRA1_P38811(3026) 3 2 4 4 1.86E-10 AEFFTLKGMFLSK(7)-KPLDCK(1):0 179  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence inter EAF7_P53911(122)-EAF3_Q12432(311)   9 5 5.43E-07 KQDFTLPWEEYGELILENAR(1)-LQYDELLKK(8):0 inter SWC4_P53201(96)-EPL1_P43572(648)   10 4 2.20E-11 SNIKYVDR(4)-HWVKGSK(4):0 inter EAF1_Q06337(784)-TRA1_P38811(2984) 1 5 5 5 7.96E-19 IYTLPNIEIQEAFLKLR(15)-KPLDCK(1):0 inter EPL1_P43572(569)-TRA1_P38811(3478) 10 11 13 10 1.31E-28 NFSTSNVPFASIASSKFQIDR(16)-KGFDPDDIQDFMADK(1):0 inter ACT1_P60010(328)-EAF1_Q06337(415)   2  3.75E-04 ENKLSDDGR(3)-VKIIAPPER(2):0 inter EAF1_Q06337(182)-TRA1_P38811(3175)   5 8 1.67E-10 RPLESTMGGEEERHEKR(16)-TTKEDFAVIQR(3):0 inter SWC4_P53201(269)-TRA1_P38811(2573)   4 4 4.56E-07 KFEMAAKR(7)-SIPKNEK(4):0 inter YAF9_P53930(211)-SWC4_P53201(463)   9 16 1.22E-17 KHVDKYEAGMSITK(1)-VDKEIDELKQK(9):0 inter EAF1_Q06337(245)-TRA1_P38811(3268) 5 7 6 9 1.80E-06 FKDPLIKNIMAK(7)-KNPK(1):0 inter EAF1_Q06337(867)-TRA1_P38811(2984)   2  2.41E-07 NGQASAPRPNQKQYTEQDIIESYSR(12)-IYTLPNIEIQEAFLKLR(15):0 inter SWC4_P53201(68)-EAF1_Q06337(406)   4  5.71E-07 EKMLSTSK(2)-KTLLPGK(1):0 inter EPL1_P43572(769)-TRA1_P38811(2713) 4 4 4 3 1.16E-05 AQQLYEVAQVKAR(11)-LKQK(2):0 inter EAF5_P39995(183)-EPL1_P43572(810) 4 3 4 4 3.54E-06 KLNDSLINSEAK(1)-EKLLK(2):0 inter SWC4_P53201(36)-ACT1_P60010(328)   7 8 2.70E-07 SPTNGQVSVPSSSAANRPKPQVTGMQR(19)-VKIIAPPER(2):0 inter SWC4_P53201(412)-EPL1_P43572(427)   2 5 8.48E-10 STKLSTFKPALQNK(3)-AAAAAAAAKAK(9):0 inter EPL1_P43572(427)-TRA1_P38811(2984)   3  1.54E-12 IYTLPNIEIQEAFLKLR(15)-AAAAAAAAKAK(9):0 inter SWC4_P53201(68)-EPL1_P43572(648)   10 14 8.91E-11 SNIKYVDR(4)-EKMLSTSK(2):0 inter SWC4_P53201(412)-EPL1_P43572(440)   3 1 9.30E-05 STKLSTFKPALQNK(3)-NNQLEDKSSR(7):0 180  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence inter YAF9_P53930(2)-TRA1_P38811(2589)    3 1.48E-05 SIITLLSKPYHTR(8)-APTISK(1):0 inter EAF1_Q06337(968)-TRA1_P38811(2984)    5 4.98E-17 IYTLPNIEIQEAFLKLR(15)-IKSPTPQEILQR(2):0 inter YNG2_P38806(199)-SWC4_P53201(412)   4 3 2.82E-06 STKLSTFKPALQNK(3)-TKEFK(2):0 inter YNG2_P38806(70)-EPL1_P43572(367)  1 2 1 7.71E-13 ENVSLNWINDELKIFDQR(13)-HPQEDGLDKEIKESLLK(9):0 inter SWC4_P53201(157)-ARP4_P80428(313)    2 3.49E-08 SNSGVVKTWR(7)-KDESK(1):0 inter EAF1_Q06337(913)-TRA1_P38811(2984)   4 5 7.28E-19 IYTLPNIEIQEAFLKLR(15)-EQQQQLKQHQIQQQR(7):0 inter YAF9_P53930(205)-SWC4_P53201(462)  5  8 8.45E-10 VDKEIDELK(3)-INTLIDLKK(8):0 inter EAF1_Q06337(803)-TRA1_P38811(2984)   3  6.19E-09 IYTLPNIEIQEAFLKLR(15)-VPTPAEMSLLKAQR(11):0 inter EAF1_Q06337(821)-TRA1_P38811(2808)    4 2.79E-06 KGDQEVR(1)-TVKNR(3):0 inter YNG2_P38806(73)-EPL1_P43572(367) 2 7 43 40 1.75E-12 ENVSLNWINDELKIFDQR(13)-HPQEDGLDKEIKESLLK(12):0 inter EAF1_Q06337(557)-EPL1_P43572(648) 2  7 3 4.18E-06 LSIFVDELNTFEKTLIQDLPLYNGINEERPK(13)-SNIKYVDR(4):0 inter EAF1_Q06337(25)-EAF5_P39995(123)  3 3 3 3.45E-08 KTQVEQIR(1)-SKFPK(2):0 inter EAF1_Q06337(713)-TRA1_P38811(2589)    4 1.65E-05 SIITLLSKPYHTR(8)-FNFSDLKGPR(7):0 inter SWC4_P53201(66)-EPL1_P43572(648) 10 9 11 8 8.08E-14 SNIKYVDR(4)-SGNNFKEK(6):0 inter EAF1_Q06337(240)-TRA1_P38811(3271) 6 10 9 12 3.32E-11 KNPKLPENTEK(4)-FKDPLIK(2):0 inter SWC4_P53201(99)-EPL1_P43572(648)    4 4.05E-07 GSKELIGDTPK(3)-SNIKYVDR(4):0 inter EAF1_Q06337(576)-EPL1_P43572(586)   4 8 2.04E-10 SFYSSHLPEYLKGISDDIR(12)-KDDSLPFIPISK(1):0 inter EAF1_Q06337(821)-TRA1_P38811(2878)  2   2.01E-06 AQEIKR(5)-TVKNR(3):0 181  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence inter EAF1_Q06337(821)-TRA1_P38811(2984) 3 3 7 7 1.37E-04 IYTLPNIEIQEAFLKLR(15)-TVKNR(3):0 inter YAF9_P53930(107)-SWC4_P53201(412) 7 29 67 41 2.81E-16 VYFVEEANEKVLNFYHR(10)-STKLSTFKPALQNK(3):0 inter EAF1_Q06337(1)-TRA1_P38811(3282)   1 1 4.65E-04 NLVKFSTTLLAPYIRPKFNADFIDNK(4)-MSSR(1):0 inter YAF9_P53930(7)-SWC4_P53201(372)    2 5.76E-04 KSESAYAEQLLK(1)-APTISKR(6):0 inter EAF6_P47128(17)-EPL1_P43572(367)   5 10 2.68E-10 ENVSLNWINDELKIFDQR(13)-KSLQDR(1):0 inter EAF1_Q06337(913)-TRA1_P38811(3161)   2  1.42E-07 EQQQQLKQHQIQQQR(7)-IAKSYPQALHFQLR(3):0 inter EAF6_P47128(104)-EPL1_P43572(395)  1 7 11 4.62E-07 IFSLSSATYVKQQHGQSQND(11)-SLNISGEDDDLINHKR(15):0 inter YNG2_P38806(39)-EPL1_P43572(395) 6 19 7 23 2.20E-15 SLNISGEDDDLINHKR(15)-LIEEKK(5):0 inter EAF1_Q06337(240)-EPL1_P43572(678)  7 10 11 1.64E-16 SACSLMDFVDFDSIEKEQYSR(16)-FKDPLIK(2):0 inter YNG2_P38806(41)-EAF6_P47128(104)  1 1  2.00E-10 IFSLSSATYVKQQHGQSQND(11)-KKYEQK(2):0 inter SWC4_P53201(238)-ARP4_P80428(326)   2 1 2.26E-06 TKPSGVNK(2)-KYLQR(1):0 inter YNG2_P38806(40)-EPL1_P43572(395)   3 4 5.64E-13 SLNISGEDDDLINHKR(15)-KKYEQK(1):0 inter EAF1_Q06337(291)-EPL1_P43572(523) 5 11 19 13 8.81E-09 IPQALPLAELKYMSQTLPLINLIPR(11)-FVQEKMEK(5):0 inter EAF1_Q06337(617)-TRA1_P38811(2589)    3 8.46E-06 QLIDEEPSISQLSKR(14)-SIITLLSKPYHTR(8):0 inter SWC4_P53201(15)-EAF1_Q06337(536)   1 1 2.25E-04 KPSSSSEVVLIQHEVAASSALIETEESKK(28)-QKSR(2):0 inter SWC4_P53201(269)-EPL1_P43572(514)   2 1 5.34E-07 KFEMAAKR(7)-EKNAR(2):0 inter YNG2_P38806(39)-EAF6_P47128(104) 9 19 20 22 1.52E-08 IFSLSSATYVKQQHGQSQND(11)-LIEEKKK(5):0 inter EPL1_P43572(440)-TRA1_P38811(2984)  4 5 1 4.37E-08 IYTLPNIEIQEAFLKLR(15)-NNQLEDKSSR(7):0 182  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence inter YNG2_P38806(199)-EPL1_P43572(427)    3 3.19E-09 AAAAAAAAKAK(9)-TKEFK(2):0 inter EAF6_P47128(104)-EPL1_P43572(397) 2 4 5 14 1.62E-15 IFSLSSATYVKQQHGQSQND(11)-KRPTIVTVEQR(1):0 inter ARP4_P80428(253)-TRA1_P38811(3268)   1 9 1.30E-04 ETLCHICPTKTLEETK(10)-KNPK(1):0 inter ARP4_P80428(210)-ACT1_P60010(315)    3 3.43E-11 MQKEITALAPSSMK(3)-EIIPLFAIKQR(9):0 inter ARP4_P80428(326)-EPL1_P43572(748)  1 2 1 1.15E-04 FGTMLGTKSYEQLR(8)-TKPSGVNK(2):0 inter EAF1_Q06337(240)-TRA1_P38811(3268) 3 3 9 8 2.86E-08 KNPKLPENTEK(1)-FKDPLIK(2):0 inter ACT1_P60010(61)-EAF1_Q06337(425)  4 3 6 9.28E-08 ISEKSGRPSDTSR(4)-DSYVGDEAQSKR(11):0 inter EAF1_Q06337(159)-TRA1_P38811(3175)   1 4 2.50E-06 ENMMDSLRPAEKTGGMWNK(12)-TTKEDFAVIQR(3):0 inter YAF9_P53930(211)-SWC4_P53201(453)   4  4.66E-09 VDKEIDELKQK(9)-QEELLKK(6):0 inter EPL1_P43572(79)-ESA1_Q08649(398)    5 3.12E-10 YYKGQHIIFLNEDILDR(3)-HLDKDELQQR(4):0 inter EAF7_P53911(77)-EAF5_P39995(216)    4 6.82E-16 SPDGSIIKNEINYEDIKNETPGSVHELQLILQK(8)-LSQSYNLEKIDEMENTYSLEATTESSR(9):0 inter SWC4_P53201(238)-ARP4_P80428(313)    2 1.03E-07 SNSGVVKTWR(7)-KYLQR(1):0 inter YNG2_P38806(51)-EPL1_P43572(395) 15 16 15 19 5.32E-17 SLNISGEDDDLINHKR(15)-ESQIHKFIR(6):0 inter YAF9_P53930(10)-SWC4_P53201(372)    3 1.45E-09 IKTLSVSRPIIYGNTAK(2)-KSESAYAEQLLK(1):0 inter EAF6_P47128(104)-ARP4_P80428(210) 1 1 1 2 2.36E-08 IFSLSSATYVKQQHGQSQND(11)-EIIPLFAIKQR(9):0 inter EAF1_Q06337(899)-TRA1_P38811(2984)    5 8.34E-12 IYTLPNIEIQEAFLKLR(15)-AAKNYYR(3):0 inter EPL1_P43572(75)-YNG2_P38806(274)  2  1 1.16E-04 IYLPNDLKHLDK(8)-IEMEKNK(5):0 inter SWC4_P53201(66)-EAF1_Q06337(404)   4  2.89E-07 DYWTYGEICCVKR(12)-SGNNFKEK(6):0 183  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence inter EAF7_P53911(142)-EAF3_Q12432(227)    6 4.21E-11 KSPNSNEEYPR(1)-ISLQIPIKLK(8):0 inter ACT1_P60010(328)-EAF1_Q06337(392)   3 1 1.84E-12 VAICTAMAQAIKDYWTYGEICCVK(12)-VKIIAPPER(2):0 inter ACT1_P60010(326)-EPL1_P43572(698)  5 8 10 5.29E-19 EGSNDTDSINVYDSKYDEFVR(15)-EITALAPSSMKVK(11):0 inter SWC4_P53201(238)-ARP4_P80428(323)  1 9 12 3.89E-10 NDYVPLKR(7)-KKYLQR(2):0 inter YNG2_P38806(40)-EAF6_P47128(104)  3 9 6 7.33E-15 IFSLSSATYVKQQHGQSQND(11)-KKYEQK(1):0 inter ARP4_P80428(323)-EPL1_P43572(748)    4 6.86E-11 FGTMLGTKSYEQLR(8)-NDYVPLKR(7):0 intra ARP4_P80428(210)-ARP4_P80428(219)   10 10 3.07E-18 EIIPLFAIKQR(9)-KTFDYEVDK(1):0 intra ARP4_P80428(218)-ARP4_P80428(227)   12 5 2.33E-09 TFDYEVDKSLYDYANNR(8)-KPEFIKK(6):0 intra ARP4_P80428(259)-ARP4_P80428(296)    2 6.06E-12 YGFAEELFLPKEDDIPANWPR(11)-TLEETKTELSSTAK(6):0 intra ARP4_P80428(313)-ARP4_P80428(323)  3 8 9 1.11E-12 SNSGVVKTWR(7)-NDYVPLKR(7):0 intra ARP4_P80428(323)-ARP4_P80428(326)   2 5 5.48E-08 NDYVPLKR(7)-TKPSGVNK(2):0 intra ARP4_P80428(332)-ARP4_P80428(336)    5 1.23E-06 TKPSGVNKSDK(8)-KVTPTEEK(1):0 intra EAF1_Q06337(102)-EAF1_Q06337(118)   1 5 5.94E-06 LKDVNLINVPNQR(2)-LSDSKMSR(5):0 intra EAF1_Q06337(118)-EAF1_Q06337(133)    4 1.45E-04 ELPENSENVSVKSESHFVPSHDNSIR(12)-LSDSKMSR(5):0 intra EAF1_Q06337(25)-EAF1_Q06337(39)    5 1.01E-05 KLTELYCVSR(1)-SKFPK(2):0 intra EAF1_Q06337(353)-EAF1_Q06337(576)   8 11 2.48E-14 FIDPWKQHNTHQNILLEEAK(6)-KDDSLPFIPISK(1):0 intra EAF1_Q06337(404)-EAF1_Q06337(406) 5 6 7 6 7.12E-16 DYWTYGEICCVKR(12)-KTLLPGK(1):0 intra EAF1_Q06337(404)-EAF1_Q06337(412)   4 5 8.84E-14 DYWTYGEICCVKR(12)-TLLPGKENK(6):0 184  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra EAF1_Q06337(406)-EAF1_Q06337(415) 3 6 8 8 4.60E-12 ENKLSDDGR(3)-KTLLPGK(1):0 intra EAF1_Q06337(406)-EAF1_Q06337(425)   1 5 1.03E-05 ISEKSGRPSDTSR(4)-KTLLPGK(1):0 intra EAF1_Q06337(412)-EAF1_Q06337(425) 1 8 5 10 5.16E-08 ISEKSGRPSDTSR(4)-TLLPGKENK(6):0 intra EAF1_Q06337(415)-EAF1_Q06337(425) 4 5 8 6 4.68E-10 ISEKSGRPSDTSR(4)-ENKLSDDGR(3):0 intra EAF1_Q06337(60)-EAF1_Q06337(63)  5 5 8 1.29E-15 LNQLLELTDENKLR(12)-KEIDAFLK(1):0 intra EAF1_Q06337(60)-EAF1_Q06337(71) 3 6 21 5 2.93E-12 LNQLLELTDENKLRK(12)-KNDIR(1):0 intra EAF1_Q06337(63)-EAF1_Q06337(71) 22 21 25 22 1.88E-12 KEIDAFLK(1)-KNDIRR(1):0 intra EAF1_Q06337(71)-EAF1_Q06337(87)  2 9 9 1.18E-07 FDEASLPKLLHTAATPITK(8)-KNDIR(1):0 intra EAF1_Q06337(71)-EAF1_Q06337(98)  1 7 5 1.59E-12 LLHTAATPITKK(11)-KNDIR(1):0 intra EAF1_Q06337(821)-EAF1_Q06337(913)   1 5 2.52E-05 EQQQQLKQHQIQQQR(7)-TVKNR(3):0 intra EAF1_Q06337(867)-EAF1_Q06337(881)  1 4 7 1.23E-11 NGQASAPRPNQKQYTEQDIIESYSR(12)-KLLEQKPDIGPEMALK(1):0 intra EAF1_Q06337(867)-EAF1_Q06337(899)   2 5 2.03E-06 NGQASAPRPNQKQYTEQDIIESYSR(12)-AAKNYYR(3):0 intra EAF1_Q06337(867)-EAF1_Q06337(913)  4 26 34 4.03E-17 PNQKQYTEQDIIESYSR(4)-EQQQQLKQHQIQQQR(7):0 intra EAF1_Q06337(899)-EAF1_Q06337(913)   4 4 6.89E-09 EQQQQLKQHQIQQQR(7)-AAKNYYR(3):0 intra EAF1_Q06337(913)-EAF1_Q06337(968)   2 3 6.37E-14 EQQQQLKQHQIQQQR(7)-IKSPTPQEILQR(2):0 intra EAF1_Q06337(98)-EAF1_Q06337(100)   1 1 4.68E-07 KLKDVNLINVPNQR(1)-LLHTAATPITKK(11):0 intra EAF1_Q06337(98)-EAF1_Q06337(102) 2 5 11 8 1.83E-28 LKDVNLINVPNQR(2)-LLHTAATPITKKK(11):0 intra EAF3_Q12432(108)-EAF3_Q12432(116) 3 8 29 29 1.13E-20 AYNEENIAMKK(10)-LANEAKEAKK(6):0 185  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra EAF3_Q12432(109)-EAF3_Q12432(116)   5  1.22E-08 AYNEENIAMKKR(11)-LANEAKEAKK(6):0 intra EAF3_Q12432(109)-EAF3_Q12432(119)   3 4 7.79E-11 AYNEENIAMKKR(11)-LANEAKEAKK(9):0 intra EAF3_Q12432(116)-EAF3_Q12432(120)    4 1.42E-12 KSLLEQQK(1)-LANEAKEAK(6):0 intra EAF3_Q12432(145)-EAF3_Q12432(156)    9 5.20E-07 SNASISKSTSQSFLTSSVSGR(7)-KGDSR(1):0 intra EAF3_Q12432(26)-EAF3_Q12432(45)   7 7 1.09E-09 CLAFHGPLMYEAKILK(13)-MYTSIPNDKPGGSSQATK(9):0 intra EAF3_Q12432(29)-EAF3_Q12432(45)   4 2 1.17E-09 MYTSIPNDKPGGSSQATK(9)-ILKIWDPSSK(3):0 intra EAF3_Q12432(311)-EAF3_Q12432(315) 6 7 14 18 1.83E-15 SSKDQKPLVPIR(3)-LQYDELLKK(8):0 intra EAF3_Q12432(312)-EAF3_Q12432(318)  5   1.85E-04 DQKPLVPIR(3)-KSSK(1):0 intra EAF3_Q12432(45)-EAF3_Q12432(57)   6 7 5.47E-07 MYTSIPNDKPGGSSQATK(9)-EIKPQK(3):0 intra EAF5_P39995(205)-EAF5_P39995(258)  2 10 11 1.04E-11 KVIGTDDWKLAR(9)-TGNFQKLFK(6):0 intra EAF5_P39995(208)-EAF5_P39995(225)   11 10 1.51E-12 NEINYEDIKNETPGSVHELQLILQK(9)-LFKSPDGSIIK(3):0 intra EAF5_P39995(208)-EAF5_P39995(250)   4  3.61E-09 LFKSPDGSIIK(3)-KVIGTDDWK(1):0 intra EAF7_P53911(173)-EAF7_P53911(186)   2 2 8.74E-06 ESPSTDLKNDNNKQEK(8)-NATIKVK(5):0 intra EAF7_P53911(178)-EAF7_P53911(186)   7 4 9.75E-14 NDNNKQEK(5)-NATIKVK(5):0 intra EAF7_P53911(2)-EAF7_P53911(63)   4 7 7.30E-13 VVHWTIVDEIR(1)-VFTAKDIWDK(5):0 intra EAF7_P53911(217)-EAF7_P53911(232)    4 1.79E-08 EVQSDEKELQR(7)-MKSTNK(2):0 intra EAF7_P53911(230)-EAF7_P53911(236)   4 12 1.83E-12 EHMSEEEQKMK(9)-STNKTAAPVRK(4):0 intra EAF7_P53911(236)-EAF7_P53911(243)   1 7 3.03E-07 STNKTAAPVR(4)-KSQR(1):0 186  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra EAF7_P53911(343)-EAF7_P53911(355)   3 5 1.06E-06 GKQIESEGGNLKK(2)-KTENK(1):0 intra EAF7_P53911(353)-EAF7_P53911(355)   5 4 1.99E-08 QIESEGGNLKK(10)-KTENK(1):0 intra EAF7_P53911(353)-EAF7_P53911(359)    2 1.82E-04 TENKKGDDQQDDTKK(4)-QIESEGGNLKKK(10):0 intra EAF7_P53911(353)-EAF7_P53911(360)    3 1.86E-06 KGDDQQDDTKK(1)-QIESEGGNLKK(10):0 intra EAF7_P53911(355)-EAF7_P53911(360)   3 7 1.26E-09 KGDDQQDDTKK(1)-KTENK(1):0 intra EAF7_P53911(360)-EAF7_P53911(375)    2 1.14E-04 KGDDQQDDTKK(1)-DKNEPLAK(2):0 intra EAF7_P53911(369)-EAF7_P53911(373)   5 4 9.21E-15 KGDDQQDDTKK(10)-DSKDKNEPLAK(3):0 intra EPL1_P43572(100)-EPL1_P43572(116)   7 3 2.45E-11 ILQMGSGHTKHK(10)-NEEKEVHLHR(4):0 intra EPL1_P43572(16)-EPL1_P43572(59)    5 4.40E-12 PTPSNAIEINDGSHKSGR(15)-KISVK(1):0 intra EPL1_P43572(39)-EPL1_P43572(59)  3 6 8 3.21E-08 SAHDDGLDSFSKGDSGAGASAGSSNSR(12)-KISVK(1):0 intra EPL1_P43572(395)-EPL1_P43572(397) 6 13 14 19 2.38E-11 SLNISGEDDDLINHKR(15)-KRPTIVTVEQR(1):0 intra EPL1_P43572(417)-EPL1_P43572(427)  4 3 6 3.00E-09 AAAAAAAAKAK(9)-KAELKR(5):0 intra EPL1_P43572(417)-EPL1_P43572(440)   4  7.64E-10 NNQLEDKSSR(7)-KAELKR(5):0 intra EPL1_P43572(427)-EPL1_P43572(432)  2   4.76E-06 AAAAAAAAKAK(9)-NNKR(3):0 intra EPL1_P43572(427)-EPL1_P43572(440) 2 4 8 9 4.39E-17 NNQLEDKSSR(7)-AAAAAAAAKAK(9):0 intra EPL1_P43572(427)-EPL1_P43572(470)   3  2.88E-08 TENGKQLANASSSSTSQPITSHVYVK(5)-AAAAAAAAKAK(9):0 intra EPL1_P43572(429)-EPL1_P43572(440)   3 2 5.07E-06 NNQLEDKSSR(7)-AKNNKR(2):0 intra EPL1_P43572(432)-EPL1_P43572(440) 1 3  4 1.95E-06 NNQLEDKSSR(7)-NNKR(3):0 187  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra EPL1_P43572(514)-EPL1_P43572(518) 3 4 8 4 1.44E-06 KFVQEK(1)-EKNAR(2):0 intra EPL1_P43572(518)-EPL1_P43572(526)   5 1 5.36E-05 KFVQEK(1)-MEKR(3):0 intra EPL1_P43572(586)-EPL1_P43572(604)   2 3 1.51E-09 SFYSSHLPEYLKGISDDIR(12)-NKDNYNLDTKR(2):0 intra EPL1_P43572(586)-EPL1_P43572(612) 9 17 29 20 5.59E-10 SFYSSHLPEYLKGISDDIR(12)-NKDNYNLDTKR(10):0 intra EPL1_P43572(748)-EPL1_P43572(759)   3 4 1.11E-06 FGTMLGTKSYEQLR(8)-EATIKYR(5):0 intra EPL1_P43572(796)-EPL1_P43572(810)  4 4 5 8.73E-19 QKSQNNNSNSSNSLK(2)-KLNDSLINSEAK(1):0 intra EPL1_P43572(809)-EPL1_P43572(821)  3 3 3 1.46E-06 LNDSLINSEAKQNSSITQK(11)-SQNNNSNSSNSLKK(13):0 intra ESA1_Q08649(24)-ESA1_Q08649(84)    5 6.05E-11 INSVDDIIIKCQCWVQK(10)-LKATDEDNKK(2):0 intra ESA1_Q08649(330)-ESA1_Q08649(432)  3 3 2 3.96E-05 LIWKPPVFTASQLR(4)-KENK(1):0 intra SWC4_P53201(125)-SWC4_P53201(204)   2  5.14E-11 FNQHLSIPSFTKEEYEAFMNENEGTQK(12)-EKFYYTCR(2):0 intra SWC4_P53201(125)-SWC4_P53201(214)    2 7.06E-13 FNQHLSIPSFTKEEYEAFMNENEGTQKSVESEK(12)-NYFKASDPSNPLLSSLNFSAEK(4):0 intra SWC4_P53201(140)-SWC4_P53201(214)   1 4 1.96E-18 EEYEAFMNENEGTQKSVESEK(15)-NYFKASDPSNPLLSSLNFSAEK(4):0 intra SWC4_P53201(146)-SWC4_P53201(157)  1 5 5 3.89E-10 SVESEKNHNENFTNEK(6)-KDESK(1):0 intra SWC4_P53201(146)-SWC4_P53201(204) 5 9 15 10 4.59E-18 SVESEKNHNENFTNEK(6)-EKFYYTCR(2):0 intra SWC4_P53201(146)-SWC4_P53201(214)   4 10 1.03E-19 NYFKASDPSNPLLSSLNFSAEK(4)-SVESEKNHNENFTNEK(6):0 intra SWC4_P53201(146)-SWC4_P53201(232)   3 2 6.97E-11 ASDPSNPLLSSLNFSAEKEIER(18)-SVESEKNHNENFTNEK(6):0 intra SWC4_P53201(146)-SWC4_P53201(238)  5 5 5 4.12E-09 SVESEKNHNENFTNEK(6)-KYLQR(1):0 intra SWC4_P53201(156)-SWC4_P53201(204)  4 17 14 4.84E-21 NHNENFTNEKKDESK(10)-EKFYYTCR(2):0 188  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra SWC4_P53201(156)-SWC4_P53201(238)   8 13 1.31E-12 NHNENFTNEKKDESK(10)-KYLQR(1):0 intra SWC4_P53201(157)-SWC4_P53201(204)  6 13 10 5.71E-15 KDESKNSWSFEEIEYLFNLCK(1)-EKFYYTCR(2):0 intra SWC4_P53201(157)-SWC4_P53201(214)    3 3.54E-07 NYFKASDPSNPLLSSLNFSAEK(4)-KDESK(1):0 intra SWC4_P53201(157)-SWC4_P53201(238)   6 4 1.32E-07 KYLQR(1)-KDESK(1):0 intra SWC4_P53201(232)-SWC4_P53201(238)   3 4 8.37E-10 ASDPSNPLLSSLNFSAEKEIER(18)-KYLQR(1):0 intra SWC4_P53201(345)-SWC4_P53201(372)   4  1.30E-08 KSESAYAEQLLK(1)-QLQQLNVKK(8):0 intra SWC4_P53201(346)-SWC4_P53201(372)    3 1.22E-06 KSESAYAEQLLK(1)-KSEVK(1):0 intra SWC4_P53201(356)-SWC4_P53201(372)   4 1 1.60E-05 KSESAYAEQLLK(1)-ENLSPKK(6):0 intra SWC4_P53201(357)-SWC4_P53201(372)  5 4 9 3.35E-08 KSESAYAEQLLK(1)-KTKR(1):0 intra SWC4_P53201(412)-SWC4_P53201(453)  4 5 3 7.87E-10 STKLSTFKPALQNK(3)-QEELLKK(6):0 intra SWC4_P53201(417)-SWC4_P53201(463)   7  1.07E-11 KHVDKYEAGMSITK(1)-LSTFKPALQNK(5):0 intra SWC4_P53201(74)-SWC4_P53201(99)   5  6.68E-09 MLSTSKPSPWSFVEFK(6)-GSKELIGDTPK(3):0 intra SWC4_P53201(84)-SWC4_P53201(107)   4 3 1.47E-10 PSPWSFVEFKANNSVTLR(10)-ELIGDTPKESPYSK(8):0 intra SWC4_P53201(96)-SWC4_P53201(107)   1 4 1.43E-06 ELIGDTPKESPYSK(8)-HWVKGSK(4):0 intra TRA1_P38811(1051)-TRA1_P38811(1232)  10 20 24 3.52E-16 KYQYNLANGLLFVLK(1)-TAVNSIKLER(7):0 intra TRA1_P38811(1169)-TRA1_P38811(1217)    5 5.47E-10 GGVLGIKVLIDNVK(7)-EVGVLAYKR(8):0 intra TRA1_P38811(1169)-TRA1_P38811(1224) 5 10 8 10 2.29E-13 VLIDNVKSSSVFLK(7)-EVGVLAYKR(8):0 intra TRA1_P38811(1613)-TRA1_P38811(1657) 3 5 10 17 7.43E-14 ELDNFYDFYISNIPKNQVR(15)-KNMTLR(1):0 189  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra TRA1_P38811(1657)-TRA1_P38811(1704)    2 1.15E-05 ELDNFYDFYISNIPKNQVR(15)-DMLNLTLKTIK(8):0 intra TRA1_P38811(1763)-TRA1_P38811(1804)   2  3.53E-05 IFVLKNVINSTLIYEVATSGSLK(5)-ASYSLKK(6):0 intra TRA1_P38811(2177)-TRA1_P38811(2193)   3  4.44E-05 EWIMENLPTIQNLLEKCIK(16)-TKEWIMENLPTIQNLLEK(2):0 intra TRA1_P38811(2309)-TRA1_P38811(2351)  4 8 3 8.79E-11 ITTKLLEK(4)-KIVNMSR(1):0 intra TRA1_P38811(2569)-TRA1_P38811(2576)    10 9.07E-10 AWVTLFPQVYKSIPK(11)-NEKYGFVR(3):0 intra TRA1_P38811(2781)-TRA1_P38811(2808)   10 9 3.29E-14 VADWNSDRDALEQSVKSVMDVPTPR(16)-KGDQEVR(1):0 intra TRA1_P38811(2795)-TRA1_P38811(2808)    9 1.62E-11 RQMFKTFLALQNFAESR(5)-KGDQEVR(1):0 intra TRA1_P38811(2795)-TRA1_P38811(2815)   8 10 2.14E-21 QMFKTFLALQNFAESR(4)-KLCDEGIQLSLIK(1):0 intra TRA1_P38811(2808)-TRA1_P38811(2815) 2  5 3 3.45E-13 KLCDEGIQLSLIK(1)-KGDQEVR(1):0 intra TRA1_P38811(2956)-TRA1_P38811(3428)   4  4.57E-08 KHNMPDVCISQLAR(1)-LFNKSLSK(4):0 intra TRA1_P38811(2956)-TRA1_P38811(3432)   15 10 1.51E-11 KHNMPDVCISQLAR(1)-SLSKNVETR(4):0 intra TRA1_P38811(3096)-TRA1_P38811(3432)    4 3.86E-05 SLSKNVETR(4)-NSKIR(3):0 intra TRA1_P38811(3175)-TRA1_P38811(3192) 1 6 9 7 2.34E-16 QTMAVMGDKPDTNDR(9)-TTKEDFAVIQR(3):0 intra TRA1_P38811(3271)-TRA1_P38811(3304)    4 5.49E-08 FNADFIDNKPDYETYIK(9)-KNPKLPENTEK(4):0 intra TRA1_P38811(3278)-TRA1_P38811(3304)  3 8 9 1.11E-12 FNADFIDNKPDYETYIK(9)-LPENTEKNLVK(7):0 intra TRA1_P38811(3282)-TRA1_P38811(3304)    5 5.38E-25 FNADFIDNKPDYETYIK(9)-NLVKFSTTLLAPYIRPK(4):0 intra TRA1_P38811(3362)-TRA1_P38811(3428)   4 3 2.83E-06 FEDIEIPGQYLLNKDNNVHFIK(14)-LFNKSLSK(4):0 intra TRA1_P38811(451)-TRA1_P38811(602) 16 22 13 10 8.27E-19 TIIHDLKVFNPPPNEYTVANPK(7)-LGKENPQEAPR(3):0 190  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra TRA1_P38811(496)-TRA1_P38811(504)   3 8 1.19E-08 LKNSIQDNDKESEEFMR(2)-YETHKK(5):0 intra TRA1_P38811(499)-TRA1_P38811(1943)  7 3 3 4.82E-06 YLVKQSLDVLTPVLHER(4)-EKAEK(2):0 intra TRA1_P38811(499)-TRA1_P38811(504)   4 5 1.01E-04 LKNSIQDNDKESEEFMR(2)-EKAEK(2):0 intra TRA1_P38811(499)-TRA1_P38811(552)  2  3 4.91E-06 KEDINDSPDVEMTESDKVVK(17)-EKAEK(2):0 intra TRA1_P38811(499)-TRA1_P38811(581)  4 2 2 2.93E-04 NYAPILLLPTPTNDPIKDAFYLYR(17)-EKAEK(2):0 intra TRA1_P38811(499)-TRA1_P38811(654)  4 3  5.48E-05 DHNEKLSPETTKK(5)-EKAEK(2):0 intra TRA1_P38811(520)-TRA1_P38811(2215)    3 2.46E-04 KVLEPSDDDHLMPQPK(1)-VLQVIMKAIK(7):0 intra TRA1_P38811(520)-TRA1_P38811(2218) 2 8 14 9 3.52E-24 AIKAQGVSVIIEEESPGK(3)-KVLEPSDDDHLMPQPK(1):0 intra TRA1_P38811(520)-TRA1_P38811(2321)  5 3 14 1.45E-17 KVLEPSDDDHLMPQPK(1)-VLYILSLKVSLLGDSR(8):0 intra TRA1_P38811(536)-TRA1_P38811(1015)  1 3 5 1.11E-05 KEDINDSPDVEMTESDK(1)-KSAYK(1):0 intra TRA1_P38811(564)-TRA1_P38811(1763)  2  2 2.31E-05 NDVEMFDIKNYAPILLLPTPTNDPIK(9)-ASYSLKK(6):0 intra TRA1_P38811(649)-TRA1_P38811(661)  2 4 4 1.39E-10 FFKDHNEK(3)-LSPETTKK(7):0 intra TRA1_P38811(74)-TRA1_P38811(197)   1 1 2.22E-04 EVPISYDAHSPEQKLR(14)-DFPSKQSSTEPR(5):0 intra TRA1_P38811(961)-TRA1_P38811(3362)  7 10 10 5.79E-13 FEDIEIPGQYLLNKDNNVHFIK(14)-QFLKPPTDLTEK(4):0 intra YAF9_P53930(2)-YAF9_P53930(60)  1 3 5 2.26E-12 GPQNEDISYFIKK(12)-APTISK(1):0 intra YNG2_P38806(194)-YNG2_P38806(199)   4  8.08E-06 TKEFK(2)-KIAR(1):0 intra YNG2_P38806(194)-YNG2_P38806(202)  3  8 8.04E-08 EFKNSR(3)-KIAR(1):0 intra YNG2_P38806(269)-YNG2_P38806(276)    7 7.91E-07 GTWYCPECKIEMEK(9)-NKLKR(2):0 191  Table A.2. Results from crosslinking mass-spectrometry of NuA4    Spectral count   Inter- or intra-molecular Cross-linked sites 40 uM 80 uM 160 uM 240 uM Best E-value Peptide sequence intra YNG2_P38806(34)-YNG2_P38806(78)   1 1 3.05E-09 YLLEEIGSNDLKLIEEK(12)-ESLLKCQSLQR(5):0 intra YNG2_P38806(41)-YNG2_P38806(51)    4 2.54E-09 ESQIHKFIR(6)-KKYEQK(2):0 intra YNG2_P38806(41)-YNG2_P38806(70)    3 1.57E-06 HPQEDGLDKEIK(9)-KKYEQK(2):0 intra YNG2_P38806(41)-YNG2_P38806(78)  3 13 9 1.30E-11 ESLLKCQSLQR(5)-KKYEQK(2):0 intra YNG2_P38806(70)-YNG2_P38806(78)   2 9 3.99E-12 HPQEDGLDKEIK(9)-ESLLKCQSLQR(5):0             192    Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide inter Elp2(147)-Elp3(543) 8 20 27 30 85 1423.03 3 4266.06192 -3.60659 3.32E-21 QNIQNDEFGLAHEFTIKK(17)-NYYGKLGYELDGPYMSK(5) inter Elp1(220)-Elp2(650) 12 14 16 17 59 756.17 4 3019.65666 3.033783 3.43E-11 EALASLKASGLVGNQLR(7)-FKNEKPHTR(5) inter Elp3(6)-Elp4(399) 7 12 19 21 59 699.07 3 2094.17379 -6.83129 9.74E-10 VLKSEWAFK(3)-ARHGKGPK(5) inter Elp1(199)-Elp4(218) 9 13 16 19 57 559.638 3 1675.88932 -2.37748 1.65E-11 KETQFR(1)-LADEKR(5) inter Elp1(311)-Elp3(311)  6 31 19 56 1119.55 5 5592.6783 -2.49808 1.85E-20 EGQLDSASEPVTGMEHQLSWKPQGSLIASIQR(21)-DIEQFKEYFENPDFR(6) inter Elp1(199)-Elp2(460) 11 13 14 17 55 722.724 3 2165.14804 -0.87047 4.46E-16 FVSGGDEKILR(8)-KETQFR(1) inter Elp1(220)-Elp2(599) 10 11 15 16 52 916.311 5 4576.50566 -3.18506 6.09E-28 IFSTENWLEIKPALPFHSLTITR(11)-EALASLKASGLVGNQLR(7) inter Elp3(6)-Elp5(140)  13 13 26 52 470.006 4 1875.99148 -2.44052 1.14E-08 DIKDENR(3)-ARHGKGPK(5) inter Elp1(220)-Elp3(468) 10 14 14 10 48 865.486 3 2593.42276 -5.20456 3.25E-10 EALASLKASGLVGNQLR(7)-KYTYR(1) inter Elp3(342)-Elp4(375)   19 26 45 1058.59 3 3171.75564 3.417857 4.99E-17 SQPGKIQHGLVHILK(5)-GTGLYELWKTGR(9) inter Elp1(220)-Elp2(647) 6 11 11 17 45 604.94 5 3019.65666 -2.70096 4.73E-17 EALASLKASGLVGNQLR(7)-FKNEKPHTR(2) inter Elp1(220)-Elp2(629) 3 6 12 19 40 714.15 4 2851.5708 1.158253 3.27E-19 EALASLKASGLVGNQLR(7)-KWALWER(1) inter Elp3(14)-Elp5(140) 3 4 16 17 40 571.313 3 1710.91519 -1.57939 2.70E-08 DIKDENR(3)-KLAPEK(1) inter Elp4(204)-Elp3(342) 5 11 11 12 39 728.729 3 2183.16263 -1.61971 1.00E-07 GTGLYELWKTGR(9)-YKDLK(2) inter Elp1(198)-Elp2(460) 6 11 11 10 38 790.438 3 2368.29027 -0.62535 3.10E-12 FVSGGDEKILR(8)-HVTVGWGKK(8) inter Elp1(199)-Elp3(342) 4 10 11 13 38 776.079 3 2325.21171 -1.04954 1.27E-21 GTGLYELWKTGR(9)-KETQFR(1) inter Elp3(84)-Elp2(250) 7 7 11 13 38 643.413 3 1927.21215 -1.90825 1.15E-09 KLTLLSNK(1)-YLLPKLK(5) inter Elp4(177)-Elp1(190)  8 13 16 37 632.096 4 2524.35367 -0.16812 3.31E-23 ISKHVTVGWGK(3)-KNLISEEESK(1) 193  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide inter Elp3(9)-Elp4(399)  6 14 16 36 674.06 3 2016.14077 -3.67777 2.20E-08 VLKSEWAFK(3)-GPKTNKK(3) inter Elp6(230)-Elp3(311)  1 20 15 36 753.165 5 3760.7831 -1.85812 6.11E-22 DIEQFKEYFENPDFR(6)-TGFAKDVTGSLHVCR(5) inter Elp3(325)-Elp1(1217) 7 9 9 10 35 653.119 4 2608.44757 -0.04248 9.15E-12 TDGLKIYPTLVIR(5)-YTGKTGGTAK(4) inter Elp1(198)-Elp3(342) 2 6 12 14 34 843.792 3 2528.35394 -0.55945 4.94E-16 GTGLYELWKTGR(9)-HVTVGWGKK(8) inter Elp2(250)-Elp1(967) 4 9 10 11 34 891.16 3 2670.45199 -2.43164 1.34E-20 KFLIDDYLGNYEK(1)-KLTLLSNK(1) inter Elp1(220)-Elp2(460) 11 9 5 6 31 1028.91 3 3083.69784 -2.3141 2.14E-17 EALASLKASGLVGNQLR(7)-FVSGGDEKILR(8) inter Elp3(342)-Elp4(370)  9 10 12 31 606.829 4 2423.28487 -1.11865 1.89E-10 GTGLYELWKTGR(9)-VYKSQPGK(3) inter Elp4(207)-Elp3(342)  8 11 11 30 797.777 3 2390.29978 -3.4555 9.00E-10 GTGLYELWKTGR(9)-DLKIAWK(3) inter Elp3(79)-Elp2(250) 9 8 7 4 28 454.539 4 1814.12809 0.278701 4.17E-12 KLTLLSNK(1)-KYLLPK(1) inter Elp2(249)-Elp1(967) 5 6 8 8 27 1125.89 3 3374.62928 -2.14512 3.85E-19 INDLIDDSEEDSKK(13)-KFLIDDYLGNYEK(1) inter Elp1(220)-Elp3(473)  5 9 13 27 690.633 4 2758.49771 -2.3739 7.99E-18 EALASLKASGLVGNQLR(7)-KEFTSQR(1) inter Elp6(225)-Elp4(399)   10 15 25 1002.56 3 3004.64595 -2.77935 1.98E-15 SMINLNLNPLKTGFAK(11)-VLKSEWAFK(3) inter Elp4(55)-Elp6(97)  1 1 22 24 1085.58 4 4338.30117 -1.15483 3.92E-20 LKIPSNNYNVLDFLSDFIVNNIHNKPR(2)-KLNIADESK(1) inter Elp3(14)-Elp4(399) 2 4 6 11 23 483.281 4 1929.0975 0.586803 5.87E-12 VLKSEWAFK(3)-KLAPEK(1) inter Elp3(325)-Elp4(451) 4 6 6 6 22 822.47 3 2464.38285 -2.29939 3.71E-09 TDGLKIYPTLVIR(5)-TKISLDY(2) inter Elp3(9)-Elp5(140)   7 10 17 600.327 3 1797.95846 -0.89602 3.34E-09 DIKDENR(3)-GPKTNKK(3) inter Elp3(61)-Elp1(846)   9 8 17 419.49 4 1670.91779 -1.85551 7.76E-08 LKQQPR(2)-KMFDPK(1) inter Elp1(198)-Elp4(218) 3 3 5 6 17 627.352 3 1879.03155 -1.48459 6.04E-10 HVTVGWGKK(8)-LADEKR(5) inter Elp4(282)-Elp1(756)  2 7 8 17 466.959 3 1397.85407 -0.05752 1.31E-06 NIMAKR(5)-KLIR(1) 194  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide inter Elp3(14)-Elp1(1270)  1 5 10 16 655.378 3 1963.1102 -0.86725 4.30E-12 LNQTKPDAVR(5)-KLAPEK(1) inter Elp1(206)-Elp2(460) 1 4 4 5 14 462.26 4 1845.01081 0.284443 6.67E-05 FVSGGDEKILR(8)-GKGAR(2) inter Elp3(9)-Elp1(1270)  2 5 7 14 642.7 3 1922.05851 -5.03148 5.90E-06 LNQTKPDAVR(5)-GPKTNK(3) inter Elp4(451)-Elp1(1270)  4 6 4 14 706.718 3 2117.13682 2.449399 1.82E-07 LNQTKPDAVR(5)-TKISLDY(2) inter Elp1(206)-Elp3(342)  2 4 7 13 502.277 4 2005.07448 -1.34519 3.34E-06 GTGLYELWKTGR(9)-GKGAR(2) inter Elp1(199)-Elp2(599) 2 2 1 8 13 1220.33 3 3657.95586 -4.06346 1.21E-12 IFSTENWLEIKPALPFHSLTITR(11)-KETQFR(1) inter Elp3(88)-Elp2(257)  3 5 5 13 480.54 4 1914.13022 6.581407 3.88E-07 LTLLSNKQYK(7)-AKPVR(2) inter Elp3(79)-Elp2(249) 3 2 5 3 13 840.444 3 2518.30538 -2.30809 1.58E-11 INDLIDDSEEDSKK(13)-KYLLPK(1) inter Elp3(14)-Elp1(1308)  2 4 6 12 735.08 3 2202.21472 -1.533 1.60E-08 ANVKEIYSISEK(4)-KLAPEK(1) inter Elp3(423)-Elp1(844)   4 8 12 919.076 5 4590.33241 -2.90752 5.20E-12 EVGIQEVHHKVQPDQVELIR(10)-SFGMEPAPLTEMQIYMKK(17) inter Elp1(206)-Elp2(650) 3 3 3 3 12 446.25 4 1780.96963 -0.88716 9.30E-05 FKNEKPHTR(5)-GKGAR(2) inter Elp4(177)-Elp3(342)   6 5 11 898.806 3 2693.39118 -1.76083 1.19E-14 GTGLYELWKTGR(9)-KNLISEEESK(1) inter Elp1(206)-Elp2(629)  2 3 6 11 378.01 5 1884.01182 0.861532 1.65E-06 DRKWALWER(3)-GKGAR(2) inter Elp4(186)-Elp3(342)  2 3 5 10 963 4 3847.95918 -2.89358 5.63E-07 NLISEEESKVTVQNLNETQR(9)-GTGLYELWKTGR(9) inter Elp3(88)-Elp2(147)   4 5 9 568.708 5 2838.50279 -0.90682 2.23E-08 QNIQNDEFGLAHEFTIKK(17)-AKPVR(2) inter Elp1(198)-Elp2(599)  1 1 7 9 644.525 6 3861.09809 -1.53826 5.72E-11 IFSTENWLEIKPALPFHSLTITR(11)-HVTVGWGKK(8) inter Elp1(206)-Elp3(473)   4 5 9 507.61 3 1519.81068 1.173963 2.99E-04 KEFTSQR(1)-GKGAR(2) inter Elp3(14)-Elp1(846)  1 3 5 9 398.23 4 1586.87418 -6.30377 1.45E-07 KMFDPK(1)-KLAPEK(1) inter Elp3(6)-Elp1(1270)  1 3 4 8 634.69 3 1901.04827 0.091528 3.91E-05 LNQTKPDAVR(5)-HGKGPK(3) 195  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide inter Elp1(199)-Elp3(347)   3 4 7 654.603 4 2614.37548 -2.79699 2.04E-05 YKSYSANALVDLVAR(2)-KETQFR(1) inter Elp3(12)-Elp5(140)   3 4 7 548.969 3 1643.88424 -0.36195 6.98E-06 DIKDENR(3)-TNKKK(3) inter Elp4(55)-Elp6(124)   3 4 7 752.764 3 2255.26239 -3.9777 1.86E-12 DKILSDVLAK(2)-KLNIADESK(1) inter Elp3(79)-Elp1(846)  2 4 1 7 416.99 4 1662.94187 8.63858 9.22E-11 KYLLPK(1)-KMFDPK(1) inter Elp3(6)-Elp1(1119)  1 1 5 7 703.04 3 2103.09349 2.513653 1.85E-09 EAVALYCKAYR(8)-HGKGPK(3) inter Elp1(206)-Elp2(647) 4 1 2  7 430.911 3 1289.70917 -0.44498 3.83E-05 FKNEK(2)-GKGAR(2) inter Elp3(9)-Elp1(1308)  2 2 2 6 721.73 3 2161.16303 -0.81677 1.28E-04 ANVKEIYSISEK(4)-GPKTNK(3) inter Elp5(267)-Elp3(342)   2 4 6 527.05 4 2104.16805 -2.25438 8.64E-06 GTGLYELWKTGR(9)-QKLAK(2) inter Elp3(6)-Elp2(460)   3 3 6 496.28 4 1980.07922 -4.1775 1.25E-07 FVSGGDEKILR(8)-HGKGPK(3) inter Elp4(448)-Elp1(1270)    6 6 947.47 6 5675.7516 -2.58788 5.16E-07 KFEIEQWGIPVDDAEGSAASEQSHSHSHSDEISHNIPAKK(39)-LNQTKPDAVR(5) inter Elp3(19)-Elp1(1308)   2 4 6 787.76 3 2359.26346 3.670785 9.25E-06 ANVKEIYSISEK(4)-LAPEKER(5) inter Elp4(55)-Elp6(95)    6 6 764.01 5 3814.98935 -6.3292 2.33E-14 SHAVLASFIHEQNYFTNSLNKLK(21)-KLNIADESK(1) inter Elp3(14)-Elp1(1062)   3 3 6 678.69 3 2033.06154 6.612731 3.20E-14 EAMGAYQSAKR(10)-KLAPEK(1) inter Elp2(257)-Elp1(943)    6 6 963.53 4 3849.09613 2.242545 2.04E-09 SALSLYDVSLALLVAQKSQMDPR(17)-LTLLSNKQYK(7) inter Elp3(473)-Elp1(510)   2 4 6 812.09 3 2430.23653 -0.68468 6.00E-15 GKHPSIVCEFPK(2)-KEFTSQR(1) inter Elp3(464)-Elp1(507)   2 4 6 787.445 3 2359.30723 -2.0372 4.78E-05 DVLIFAAVPSIEEMKK(15)-KASK(1) inter Elp1(206)-Elp3(347)    5 5 765.753 3 2294.23825 0.609832 2.01E-05 YKSYSANALVDLVAR(2)-GKGAR(2) inter Elp3(311)-Elp1(383)    5 5 799.598 5 3992.94134 -2.58055 7.66E-14 DIEQFKEYFENPDFR(6)-IQLWTSKNYHWYLK(7) inter Elp3(6)-Elp2(370)   2 3 5 804.4 3 2410.18854 4.33329 1.27E-05 MWATKDNIICDQR(5)-HGKGPK(3) 196  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide inter Elp6(230)-Elp4(399)    5 5 723.884 4 2891.50033 -2.14421 6.24E-09 TGFAKDVTGSLHVCR(5)-VLKSEWAFK(3) inter Elp3(2)-Elp1(1270)   2 3 5 710.74 3 2128.18649 -3.901 2.03E-06 LNQTKPDAVR(5)-ARHGKGPK(1) inter Elp3(9)-Elp1(1062)    4 4 665.012 3 1992.00985 -1.67745 6.49E-09 EAMGAYQSAKR(10)-GPKTNK(3) inter Elp3(347)-Elp4(375)    4 4 693.192 5 3460.91941 -1.71674 3.45E-13 YKSYSANALVDLVAR(2)-SQPGKIQHGLVHILK(5) inter Elp4(55)-Elp1(756)    4 4 672.383 3 2014.12448 -0.58462 3.38E-10 KLNIADESK(1)-KNIMAKR(6) inter Elp3(59)-Elp1(846)    4 4 364.209 4 1452.80505 -1.17483 2.88E-04 KMFDPK(1)-YKLK(2) inter Elp3(6)-Elp1(1062)  1  3 4 494.01 4 1970.99961 -3.99896 4.75E-10 EAMGAYQSAKR(10)-HGKGPK(3) inter Elp3(84)-Elp1(846)   2 2 4 593.017 3 1776.02593 -1.66861 5.30E-08 YLLPKLK(5)-KMFDPK(1) inter Elp3(86)-Elp2(257)   2 2 4 451.779 4 1803.08696 -0.36249 4.18E-04 LTLLSNKQYK(7)-LKAK(2) inter Elp5(211)-Elp4(375)    4 4 448.667 5 2238.29604 -0.71684 5.24E-05 SQPGKIQHGLVHILK(5)-KSGR(1) inter Elp3(342)-Elp4(451)    3 3 786.419 3 2356.23144 -2.10166 1.21E-07 GTGLYELWKTGR(9)-TKISLDY(2) inter Elp3(19)-Elp1(1217)    3 3 655.36 3 1962.04218 -6.42671 2.45E-08 YTGKTGGTAK(4)-LAPEKER(5) inter Elp4(177)-Elp5(211)    3 3 440.99 4 1759.93158 0.445472 2.14E-04 KNLISEEESK(1)-KSGR(1) inter Elp5(267)-Elp1(846)    3 3 373.217 4 1488.83741 0.394939 3.10E-05 KMFDPK(1)-QKLAK(2) inter Elp1(220)-Elp3(342)    3 3 1082.26 3 3243.76151 -2.07999 3.46E-16 EALASLKASGLVGNQLR(7)-GTGLYELWKTGR(9) inter Elp3(6)-Elp1(1308)   3  3 715.06 3 2140.15279 0.604064 1.41E-04 ANVKEIYSISEK(4)-HGKGPK(3) inter Elp4(213)-Elp1(857)   2 1 3 995.029 4 3973.09822 4.94154 3.06E-04 VNKICDAVLNVLLSNPEYK(3)-YKLADEKRLGSPDR(2) inter Elp4(211)-Elp6(265)    3 3 823.63 5 4109.09968 -0.14976 1.31E-06 GGAPIATSNTSLHVVENEYLYLNEKESTK(25)-IAWKYK(4) inter Elp3(521)-Elp1(759)   2 1 3 653.09 4 2605.33221 4.33764 4.00E-05 RYKEAFIVCR(3)-IAKEEHGSEK(3) 197  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide inter Elp3(79)-Elp2(257)    3 3 559.344 4 2233.34496 -0.85629 2.52E-06 KLTLLSNKQYK(8)-KYLLPK(1) inter Elp3(19)-Elp1(846)    2 2 582.315 3 1743.92292 -0.41607 2.58E-04 LAPEKER(5)-KMFDPK(1) inter Elp3(6)-Elp2(764)   1 1 2 397.478 4 1585.88411 0.050445 3.07E-04 WSHLKR(5)-HGKGPK(3) inter Elp4(173)-Elp2(260)   2  2 658.35 5 3282.68057 -6.00174 2.80E-05 KQMKKNLISEEESK(1)-QYKFQIDDELR(3) inter Elp3(6)-Elp4(370)    2 2 556.315 3 1665.92022 -2.03791 5.10E-05 VYKSQPGK(3)-HGKGPK(3) inter Elp3(2)-Elp5(140)   1 1 2 626.338 3 1875.99148 0.141312 7.90E-06 DIKDENR(3)-ARHGKGPK(1) inter Elp3(9)-Elp2(460)    2 2 668.37 3 2001.08946 2.329568 2.54E-04 FVSGGDEKILR(8)-GPKTNK(3) inter Elp3(61)-Elp4(399)    2 2 672.39 3 2013.14111 -1.83005 8.91E-09 VLKSEWAFK(3)-LKQQPR(2) inter Elp4(177)-Elp1(187)    2 2 898.47 4 3587.84977 1.552711 4.47E-11 LFEPISEYHLEVDDLKISK(16)-KNLISEEESK(1) inter Elp4(204)-Elp1(1062)    2 2 672.348 3 2014.01935 -1.08177 4.38E-06 EAMGAYQSAKR(10)-YKDLK(2) inter Elp4(451)-Elp1(1308)   1 1 2 786.422 3 2356.24134 -1.21066 7.87E-06 ANVKEIYSISEK(4)-TKISLDY(2) inter Elp4(207)-Elp1(1223)    2 2 673.04 3 2016.10036 1.122961 8.35E-07 TGGTAKTGASR(6)-DLKIAWK(3) intra Elp2(677)-Elp2(699) 35 37 47 41 160 588.114 5 2935.52906 -1.71638 1.87E-07 HQKEPADDYVLEASIKHTK(16)-DKTVK(2) intra Elp1(759)-Elp1(875) 14 31 52 62 159 966.02 4 3858.03894 -1.36164 6.66E-21 KYLQTIITAYASQNPQNLSAALK(1)-YKEAFIVCR(2) intra Elp2(680)-Elp2(699) 24 39 49 45 157 914.491 3 2740.44354 -2.37724 1.95E-16 EPADDYVLEASIKHTK(13)-TVKVWR(3) intra Elp2(629)-Elp2(650) 16 25 26 29 96 570.3 4 2277.17666 2.575125 4.47E-15 DRKWALWER(3)-NEKPHTR(3) intra Elp1(967)-Elp1(989)  24 32 37 93 1276.88 4 5100.46548 -3.02068 9.55E-30 ALEHLSEIDKDGNVSEEVIDYVESHDLYK(10)-KFLIDDYLGNYEK(1) intra Elp2(629)-Elp2(647) 11 22 27 32 92 761.412 3 2281.21198 -0.76372 1.21E-17 FKNEKPHTR(2)-KWALWER(1) intra Elp2(650)-Elp2(680) 23 29 22 16 90 694.725 3 2081.15341 0.538211 1.30E-12 FKNEKPHTR(5)-TVKVWR(3) 198  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp3(401)-Elp3(423)  12 27 29 68 893.716 4 3570.82862 -1.9433 1.53E-25 EVGIQEVHHKVQPDQVELIR(10)-MKDLGTTCR(2) intra Elp1(1217)-Elp1(1270) 5 12 22 26 65 964.85 3 2889.54204 7.104894 2.03E-12 YTGKTGGTAKTGASRR(4)-LNQTKPDAVR(5) intra Elp1(846)-Elp1(1308) 13 17 16 17 63 761.738 3 2282.1868 -1.8317 5.91E-11 ANVKEIYSISEK(4)-KMFDPK(1) intra Elp1(198)-Elp1(220) 7 13 19 19 58 959.211 3 2874.60791 -1.55186 1.29E-21 EALASLKASGLVGNQLR(7)-HVTVGWGKK(8) intra Elp1(1232)-Elp1(1270) 3 12 17 21 53 666.049 3 1995.12251 -1.31902 3.80E-08 LNQTKPDAVR(5)-TAKNKR(3) intra Elp1(851)-Elp1(1308) 8 11 16 18 53 824.431 3 2470.26651 -2.28906 1.35E-08 ANVKEIYSISEK(4)-MFDPKTSK(5) intra Elp4(204)-Elp4(213)  6 21 24 51 609.34 3 1824.99853 0.169589 5.40E-07 YKLADEKR(2)-YKDLK(2) intra Elp3(342)-Elp3(347) 4 10 15 21 50 1063.23 3 3186.67131 -2.5274 4.09E-21 YKSYSANALVDLVAR(2)-GTGLYELWKTGR(9) intra Elp1(199)-Elp1(220) 11 12 13 11 47 891.496 3 2671.46568 -0.29991 5.42E-17 EALASLKASGLVGNQLR(7)-KETQFR(1) intra Elp2(249)-Elp2(260) 6 11 14 16 47 1071.52 3 3211.54081 -2.67192 1.64E-12 INDLIDDSEEDSKK(13)-QYKFQIDDELR(3) intra Elp1(190)-Elp1(199)  14 14 17 45 720.07 3 2156.1742 -4.91195 6.34E-13 ISKHVTVGWGK(3)-KETQFR(1) intra Elp2(619)-Elp2(686) 7 13 8 15 43 794.411 4 3173.60664 -2.04738 2.37E-23 HQKEPADDYVLEASIK(3)-DGKFLLSVCR(3) intra Elp5(47)-Elp5(100)  2 19 21 42 1095.62 3 3283.81798 -2.22393 2.79E-23 QIISYLPAATATQAKK(15)-LIQEFVHQSKSK(10) intra Elp1(187)-Elp1(199)  1 21 18 40 1074.23 3 3219.6703 -2.36514 1.74E-20 LFEPISEYHLEVDDLKISK(16)-KETQFR(1) intra Elp3(79)-Elp3(325) 5 12 11 11 39 597.615 4 2386.42392 -2.68719 4.50E-10 TDGLKIYPTLVIR(5)-KYLLPK(1) intra Elp3(9)-Elp3(19)  7 13 18 38 470.78 4 1879.08907 -1.62632 5.34E-11 KLAPEKER(6)-GPKTNKK(3) intra Elp3(84)-Elp3(88) 8 7 11 12 38 528.008 3 1581.00176 -0.40417 1.25E-07 YLLPKLK(5)-AKPVR(2) intra Elp3(311)-Elp3(325) 5 8 11 14 38 901.47 4 3601.8344 -4.5521 9.93E-23 DIEQFKEYFENPDFR(6)-TDGLKIYPTLVIR(5) intra Elp3(14)-Elp3(61) 5 15 8 9 37 531.323 3 1590.94571 -0.76954 8.44E-10 LKQQPR(2)-KLAPEK(1) 199  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp2(250)-Elp2(260) 5 10 11 11 37 627.849 4 2507.36352 -1.62752 5.26E-16 QYKFQIDDELR(3)-KLTLLSNK(1) intra Elp1(818)-Elp1(844)  11 12 14 37 882.446 4 3525.74852 -2.2959 6.34E-17 SFGMEPAPLTEMQIYMKK(17)-YKETLYSGISK(2) intra Elp4(169)-Elp4(177) 4 5 15 13 37 812.44 3 2432.27984 -4.78824 1.01E-12 KNLISEEESK(1)-ELPGIYKGSR(7) intra Elp3(502)-Elp3(543)   12 23 35 1386 3 4154.97575 -2.8751 7.32E-30 KFQHQGFGTLLMEEAER(1)-NYYGKLGYELDGPYMSK(5) intra Elp3(19)-Elp3(61) 7 4 13 11 35 584.68 3 1747.99445 -7.85029 1.43E-08 LAPEKER(5)-LKQQPR(2) intra Elp3(6)-Elp3(14)  11 11 13 35 482.622 3 1444.84017 -1.68323 4.27E-09 KLAPEK(1)-HGKGPK(3) intra Elp3(325)-Elp3(342)  6 15 13 34 1002.9 3 3005.65895 -2.15593 6.08E-19 TDGLKIYPTLVIR(5)-GTGLYELWKTGR(9) intra Elp3(289)-Elp3(325)  4 13 17 34 993.769 4 3971.03584 -2.77297 1.44E-26 DAGYKVVSHMMPDLPNVGMER(5)-TDGLKIYPTLVIR(5) intra Elp1(1062)-Elp1(1232)  1 10 23 34 690.03 3 2065.07385 5.976581 9.55E-14 EAMGAYQSAKR(10)-TAKNKR(3) intra Elp4(143)-Elp4(282)  7 12 14 33 583.33 3 1744.9683 3.894748 2.04E-10 ISDSSADKTR(8)-KLIR(1) intra Elp2(460)-Elp2(599)  9 11 13 33 1018.56 4 4070.18802 -2.22004 1.79E-05 IFSTENWLEIKPALPFHSLTITR(11)-FVSGGDEKILR(8) intra Elp3(6)-Elp3(19)  7 12 14 33 577.67 3 1729.98387 -1.73267 5.86E-09 KLAPEKER(6)-HGKGPK(3) intra Elp2(249)-Elp2(257) 4 9 10 9 32 989.182 3 2964.5179 -2.22687 1.08E-27 INDLIDDSEEDSKK(13)-LTLLSNKQYK(7) intra Elp2(2)-Elp2(768) 8 5 11 6 30 1150.61 3 3448.79114 -1.71364 1.72E-22 VECITPEAIFIGANK(1)-NGKLFLGVGSSDLSTR(3) intra Elp3(88)-Elp3(325) 8 5 7 9 29 549.833 4 2195.30414 1.003962 2.46E-05 TDGLKIYPTLVIR(5)-AKPVR(2) intra Elp1(1119)-Elp1(1232) 1 7 9 12 29 550.3 4 2197.16773 -2.09142 9.09E-10 EAVALYCKAYR(8)-TAKNKR(3) intra Elp3(12)-Elp3(19) 2 7 10 9 28 400.237 4 1596.91989 0.220174 3.90E-07 LAPEKER(5)-TNKKK(3) intra Elp3(423)-Elp3(453)   7 21 28 1177.68 3 3529.99837 -2.06459 8.65E-26 EVGIQEVHHKVQPDQVELIR(10)-KDILIGLLR(1) intra Elp1(1119)-Elp1(1223) 4 11 7 6 28 622.573 4 2486.25872 -1.30734 1.78E-10 EAVALYCKAYR(8)-TGGTAKTGASR(6) 200  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp3(464)-Elp3(473) 2 4 8 12 26 489.272 3 1464.79364 0.098103 2.28E-05 KEFTSQR(1)-KASK(1) intra Elp4(55)-Elp4(143)  7 11 8 26 745.39 3 2233.14375 -1.72165 1.33E-12 ISDSSADKTR(8)-KLNIADESK(1) intra Elp2(49)-Elp2(768)  7 9 10 26 1009.04 4 4030.14147 4.284087 7.39E-22 TIALWDPIEPNNKGVYATLK(13)-NGKLFLGVGSSDLSTR(3) intra Elp3(423)-Elp3(502)   3 23 26 1128.58 4 4510.30665 -0.71702 8.22E-39 EVGIQEVHHKVQPDQVELIR(10)-KFQHQGFGTLLMEEAER(1) intra Elp4(63)-Elp4(281)  3 12 10 25 640.36 3 1915.03744 -5.60776 2.04E-09 LNIADESKTK(8)-RNDKK(4) intra Elp4(204)-Elp4(218)  4 11 10 25 512.288 3 1533.84024 -0.39756 5.06E-09 LADEKR(5)-YKDLK(2) intra Elp4(177)-Elp4(213)  3 8 14 25 584.814 4 2335.22708 -0.11733 5.13E-10 KNLISEEESK(1)-YKLADEKR(2) intra Elp1(845)-Elp1(1308) 3 9 6 7 25 805.1 3 2410.28176 4.254899 5.97E-10 ANVKEIYSISEK(4)-KKMFDPK(1) intra Elp4(207)-Elp4(213)  1 6 17 24 626.352 3 1876.03457 -0.50788 2.37E-08 DLKIAWK(3)-YKLADEK(2) intra Elp4(204)-Elp4(211)  9 6 9 24 537.976 3 1610.9072 -0.15494 1.11E-06 IAWKYK(4)-YKDLK(2) intra Elp3(9)-Elp3(14) 3 5 5 11 24 532.323 3 1593.94537 -1.24734 1.51E-13 GPKTNKK(3)-KLAPEK(1) intra Elp4(177)-Elp4(375)  1 10 13 24 742.917 4 2967.63928 -0.02197 2.94E-17 SQPGKIQHGLVHILK(5)-KNLISEEESK(1) intra Elp1(1223)-Elp1(1270) 2 5 6 10 23 762.413 3 2284.2135 -1.35837 6.45E-12 LNQTKPDAVR(5)-TGGTAKTGASR(6) intra Elp4(177)-Elp4(218)  4 9 10 23 682.365 3 2044.06879 -2.00634 5.14E-11 KNLISEEESK(1)-LADEKR(5) intra Elp1(1119)-Elp1(1217) 1 5 7 9 22 822.76 3 2463.24676 -1.9211 9.58E-10 EAVALYCKAYR(8)-YTGKTGGTAK(4) intra Elp3(88)-Elp3(502)   9 13 22 546.492 5 2727.41662 -3.3473 1.01E-09 KFQHQGFGTLLMEEAER(1)-AKPVR(2) intra Elp1(814)-Elp1(818)  4 8 10 22 947.234 4 3784.90083 -1.13419 2.27E-19 VDYLNLFISCLSEDDVTKTK(18)-YKETLYSGISK(2) intra Elp3(12)-Elp3(61) 4 5 4 9 22 466.281 3 1395.8198 0.026221 1.60E-05 LKQQPR(2)-TNKK(3) intra Elp4(143)-Elp4(281)  8 6 7 21 470.5 4 1875.96501 0.433447 1.35E-10 ISDSSADKTR(8)-RNDKK(4) 201  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp4(169)-Elp4(235)  6 6 8 20 1128.8 4 4511.16291 -2.60996 2.09E-13 LGSPDRDDIQQNSEYKDYNHQFDITTR(16)-ELPGIYKGSR(7) intra Elp4(173)-Elp4(177)   7 13 20 463.5 4 1846.97101 5.467113 4.02E-06 KNLISEEESK(1)-KQMK(1) intra Elp2(650)-Elp2(699)   12 7 19 778.66 4 3108.58797 -5.21634 1.12E-10 EPADDYVLEASIKHTK(13)-FKNEKPHTR(5) intra Elp1(199)-Elp1(206) 1 5 6 7 19 479.6 3 1432.77865 7.281069 5.10E-07 KETQFR(1)-GKGAR(2) intra Elp1(311)-Elp1(383)    18 18 1100.16 5 5495.7612 -1.19838 6.09E-14 EGQLDSASEPVTGMEHQLSWKPQGSLIASIQR(21)-IQLWTSKNYHWYLK(7) intra Elp2(469)-Elp2(486) 1   17 18 1113.83 4 4449.28509 0.200841 1.35E-12 FVGIQFEEKSEMPDSATVPVLGLSNK(9)-SFDLPKGVAGMLQK(6) intra Elp4(143)-Elp4(169)  4 5 9 18 779.409 3 2335.20193 -1.32853 7.13E-13 ELPGIYKGSR(7)-ISDSSADKTR(8) intra Elp4(169)-Elp4(173)  9 3 5 17 597.666 3 1789.97603 -0.85526 2.75E-09 ELPGIYKGSR(7)-KQMK(1) intra Elp4(177)-Elp4(204)   4 12 16 495.769 4 1979.04627 -0.96693 2.96E-07 KNLISEEESK(1)-YKDLK(2) intra Elp4(211)-Elp4(218) 2 4 5 5 16 559.651 3 1675.92972 -0.46374 9.84E-11 IAWKYK(4)-LADEKR(5) intra Elp1(1223)-Elp1(1308)  3 7 6 16 842.45 3 2523.31802 -2.68428 6.63E-11 ANVKEIYSISEK(4)-TGGTAKTGASR(6) intra Elp3(79)-Elp3(88) 3 2 4 7 16 368.49 4 1467.9177 -4.41926 1.58E-08 KYLLPK(1)-AKPVR(2) intra Elp3(468)-Elp3(473)  3 6 7 16 588.64 3 1761.90499 5.782524 5.12E-12 KEFTSQR(1)-KYTYR(1) intra Elp3(9)-Elp3(61)  3 7 5 15 388.481 4 1549.89402 0.265309 1.37E-05 LKQQPR(2)-GPKTNK(3) intra Elp1(507)-Elp1(724)    15 15 1323.42 3 3967.23186 -3.28011 1.09E-19 GSILVSVIPSKSSVVLQATR(11)-DVLIFAAVPSIEEMKK(15) intra Elp3(6)-Elp3(61)  2 6 6 14 511.64 3 1528.88378 -2.8827 4.77E-06 LKQQPR(2)-HGKGPK(3) intra Elp1(1223)-Elp1(1232)  1 6 7 14 621.015 3 1860.01771 -2.79195 3.29E-09 TGGTAKTGASR(6)-TAKNKR(3) intra Elp1(187)-Elp1(206)  3 5 6 14 580.914 5 2899.53307 0.276941 1.32E-06 LFEPISEYHLEVDDLKISK(16)-GKGAR(2) intra Elp1(187)-Elp1(198)  2 4 8 14 685.571 5 3422.81253 -1.47437 1.39E-10 LFEPISEYHLEVDDLKISK(16)-HVTVGWGKK(8) 202  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp4(448)-Elp4(451)   3 11 14 1075.72 5 5373.57012 -2.61204 2.45E-10 KFEIEQWGIPVDDAEGSAASEQSHSHSHSDEISHNIPAKK(39)-TKISLDY(2) intra Elp3(173)-Elp3(543)  1 2 10 13 1172.09 4 4684.2943 -3.63084 2.00E-22 QLGHSIDKVEYVLMGGTFMSLPK(8)-NYYGKLGYELDGPYMSK(5) intra Elp3(473)-Elp3(528)   7 6 13 705.37 4 2814.45115 3.63946 1.07E-17 EEHGSEKISVISGVGVR(7)-KEFTSQR(1) intra Elp4(55)-Elp4(282)  2 5 6 13 421.756 4 1682.99305 -0.06892 1.26E-08 KLNIADESK(1)-KLIR(1) intra Elp4(186)-Elp4(204)   6 7 13 1045.55 3 3133.61427 -1.90674 1.10E-05 NLISEEESKVTVQNLNETQR(9)-YKDLK(2) intra Elp1(206)-Elp1(281)  1 5 7 13 376.9 3 1127.67748 -0.11661 8.52E-06 SIKR(3)-GKGAR(2) intra Elp3(12)-Elp3(59) 1 3 5 4 13 393.576 3 1177.70706 0.527381 2.73E-05 YKLK(2)-TNKK(3) intra Elp3(78)-Elp3(325)  4 5 4 13 819.216 4 3272.82714 -2.26299 1.58E-12 LTDIINSIPDQYKK(13)-TDGLKIYPTLVIR(5) intra Elp3(401)-Elp3(453)   3 10 13 753.759 3 2258.24901 -3.29918 2.08E-14 MKDLGTTCR(2)-KDILIGLLR(1) intra Elp1(844)-Elp1(851)   3 10 13 798.645 4 3190.54626 -1.96819 1.55E-11 SFGMEPAPLTEMQIYMKK(17)-MFDPKTSK(5) intra Elp1(1062)-Elp1(1223)  1 4 8 13 786.73 3 2354.16484 2.818636 5.22E-10 EAMGAYQSAKR(10)-TGGTAKTGASR(6) intra Elp3(104)-Elp3(325)   5 7 12 831.467 4 3321.83045 -2.69465 3.27E-16 TASGIAVVAVMCKPHR(13)-TDGLKIYPTLVIR(5) intra Elp4(55)-Elp4(281)   6 6 12 606.34 3 1813.98976 -0.95595 4.37E-07 KLNIADESK(1)-RNDKK(4) intra Elp1(1030)-Elp1(1052)   4 8 12 974.688 5 4868.38632 -3.16572 8.07E-12 QNVIYNIYAKHLSSNQMYTDAAVAYEMLGK(10)-LKEAMGAYQSAK(2) intra Elp4(177)-Elp4(235)    12 12 1143.8 4 4568.15789 -0.6576 4.76E-29 LGSPDRDDIQQNSEYKDYNHQFDITTR(16)-KNLISEEESK(1) intra Elp3(61)-Elp3(401)   3 8 11 664.36 3 1987.03429 -6.98774 2.10E-09 MKDLGTTCR(2)-LKQQPR(2) intra Elp4(173)-Elp4(235)  2 3 6 11 1310.63 3 3925.85408 -1.04763 1.64E-07 LGSPDRDDIQQNSEYKDYNHQFDITTR(16)-KQMK(1) intra Elp3(79)-Elp3(423)   3 8 11 651.169 5 3250.80773 -0.56817 6.84E-09 EVGIQEVHHKVQPDQVELIR(10)-KYLLPK(1) intra Elp1(199)-Elp1(311)    11 11 1107.31 4 4424.20702 -0.10757 4.54E-13 EGQLDSASEPVTGMEHQLSWKPQGSLIASIQR(21)-KETQFR(1) 203  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp1(818)-Elp1(846)  1 5 5 11 731.051 3 2190.12823 -0.96547 3.67E-11 YKETLYSGISK(2)-KMFDPK(1) intra Elp1(844)-Elp1(846)   5 6 11 751.625 4 3002.46655 -1.54379 3.23E-13 SFGMEPAPLTEMQIYMKK(17)-KMFDPK(1) intra Elp4(143)-Elp4(235)   4 6 10 1119.52 4 4471.07998 8.735708 3.18E-09 LGSPDRDDIQQNSEYKDYNHQFDITTR(16)-ISDSSADKTR(8) intra Elp1(756)-Elp1(759)  3 3 4 10 685.702 3 2054.0805 -1.6103 2.36E-07 YKEAFIVCR(2)-NIMAKR(5) intra Elp1(816)-Elp1(844)   3 7 10 926.473 3 2776.38896 -2.88547 1.47E-06 SFGMEPAPLTEMQIYMKK(17)-TKYK(2) intra Elp4(169)-Elp4(211)   3 7 10 689.055 3 2064.14077 -0.90595 4.96E-06 ELPGIYKGSR(7)-IAWKYK(4) intra Elp1(851)-Elp1(857)   2 8 10 820.683 4 3278.69303 -3.05889 6.00E-08 VNKICDAVLNVLLSNPEYK(3)-MFDPKTSK(5) intra Elp3(88)-Elp3(423) 2 4 1 3 10 765.93 4 3059.68795 -0.93016 4.42E-08 EVGIQEVHHKVQPDQVELIR(10)-AKPVR(2) intra Elp3(61)-Elp3(325)  2 4 4 10 799.142 3 2394.39984 -1.52426 6.69E-09 TDGLKIYPTLVIR(5)-LKQQPR(2) intra Elp2(677)-Elp2(702) 1 3 2 4 10 837.13 3 2506.35772 -1.50536 1.26E-04 HTKAVTAISIHDSMIR(3)-DKTVK(2) intra Elp4(169)-Elp4(186)   1 9 10 1197.29 3 3586.84784 1.767698 1.03E-09 NLISEEESKVTVQNLNETQR(9)-ELPGIYKGSR(7) intra Elp1(1052)-Elp1(1074)    9 9 968.689 5 4838.40337 -1.07907 2.66E-19 EAMSIAVQKFPEEVESVAEELISSLTFEHR(9)-LKEAMGAYQSAK(2) intra Elp3(19)-Elp3(342)  1 4 4 9 787.427 3 2359.25356 -1.71859 4.57E-13 GTGLYELWKTGR(9)-LAPEKER(5) intra Elp4(169)-Elp4(218)   4 5 9 663.366 3 1987.07381 -0.59333 1.34E-05 ELPGIYKGSR(7)-LADEKR(5) intra Elp3(467)-Elp3(473)  1 4 4 9 531.97 3 1592.8886 -0.3665 2.85E-05 KEFTSQR(1)-KASKK(4) intra Elp3(464)-Elp3(468) 1 4 2 1 8 434.247 3 1299.71869 -0.25744 1.18E-04 KYTYR(1)-KASK(1) intra Elp2(25)-Elp2(28)   4 4 8 771.11 3 2309.31068 2.546846 1.39E-14 QTQVSDIHKVK(9)-KIVAFGAGK(1) intra Elp3(9)-Elp3(59)   1 7 8 444.934 3 1331.78128 -0.13253 1.96E-06 GPKTNK(3)-YKLK(2) intra Elp4(177)-Elp4(207)   4 4 8 729.738 3 2186.18342 -3.51214 2.05E-08 KNLISEEESK(1)-DLKIAWK(3) 204  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp3(61)-Elp3(423)   7 1 8 653.16 5 3258.78365 8.204827 4.62E-08 EVGIQEVHHKVQPDQVELIR(10)-LKQQPR(2) intra Elp4(143)-Elp4(177)  2 3 3 8 798.74 3 2392.19691 0.883337 3.54E-18 KNLISEEESK(1)-ISDSSADKTR(8) intra Elp3(19)-Elp3(401)   4 4 8 688.02 3 2060.03942 2.24353 9.41E-09 MKDLGTTCR(2)-LAPEKER(5) intra Elp3(14)-Elp3(59)  1 4 3 8 501.99 3 1500.92793 -9.02626 2.35E-07 KLAPEK(1)-KYKLK(3) intra Elp1(989)-Elp1(1020)    8 8 1067.12 5 5330.567 -1.96433 6.03E-22 ALEHLSEIDKDGNVSEEVIDYVESHDLYK(10)-YDSEKQNVIYNIYAK(5) intra Elp3(56)-Elp3(59)  1 2 5 8 405.245 3 1212.71181 -0.36851 2.35E-05 YKLK(2)-YSKK(3) intra Elp1(846)-Elp1(857)   3 4 7 773.661 4 3090.61332 -0.98349 1.24E-11 VNKICDAVLNVLLSNPEYK(3)-KMFDPK(1) intra Elp4(143)-Elp4(173)   2 5 7 584.306 3 1749.8931 -0.88302 1.26E-09 ISDSSADKTR(8)-KQMK(1) intra Elp1(814)-Elp1(846)   2 5 7 816.414 4 3261.61886 -2.35429 8.17E-11 VDYLNLFISCLSEDDVTKTK(18)-KMFDPK(1) intra Elp4(63)-Elp4(143)    7 7 779.07 3 2334.19143 1.428334 1.43E-13 LNIADESKTK(8)-ISDSSADKTR(8) intra Elp1(756)-Elp1(1223)   4 3 7 626.01 3 1874.99962 -4.52051 2.95E-08 TGGTAKTGASR(6)-NIMAKR(5) intra Elp1(190)-Elp1(220)    7 7 1025.92 3 3074.724 -1.6086 1.91E-18 EALASLKASGLVGNQLR(7)-ISKHVTVGWGK(3) intra Elp4(169)-Elp4(204)   1 6 7 481.77 4 1922.05129 1.972907 3.09E-07 ELPGIYKGSR(7)-YKDLK(2) intra Elp2(486)-Elp2(764)  1 4 1 6 947.245 4 3784.94978 -0.66992 1.17E-06 FVGIQFEEKSEMPDSATVPVLGLSNK(9)-WSHLKR(5) intra Elp1(198)-Elp1(206)  1 4 1 6 546.31 3 1635.92088 7.81462 1.23E-05 HVTVGWGKK(8)-GKGAR(2) intra Elp3(6)-Elp3(342)   4 2 6 714.388 3 2140.14289 -0.22139 1.34E-06 GTGLYELWKTGR(9)-HGKGPK(3) intra Elp3(88)-Elp3(453)    6 6 437.785 4 1747.10834 -0.63602 2.50E-08 KDILIGLLR(1)-AKPVR(2) intra Elp3(325)-Elp3(347)    6 6 824.714 4 3294.82272 -1.24328 2.89E-17 YKSYSANALVDLVAR(2)-TDGLKIYPTLVIR(5) intra Elp3(173)-Elp3(502)    6 6 942.479 5 4707.35387 -1.43424 2.07E-25 QLGHSIDKVEYVLMGGTFMSLPK(8)-KFQHQGFGTLLMEEAER(1) 205  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp6(230)-Elp6(265)    6 6 990.9 5 4948.45522 -1.00536 5.04E-07 GGAPIATSNTSLHVVENEYLYLNEKESTK(25)-TGFAKDVTGSLHVCR(5) intra Elp3(325)-Elp3(423)   1 4 5 796.85 5 3978.19417 -4.02743 5.08E-09 EVGIQEVHHKVQPDQVELIR(10)-TDGLKIYPTLVIR(5) intra Elp3(56)-Elp3(61)   1 4 5 477.95 3 1430.82455 -1.64926 1.10E-04 LKQQPR(2)-YSKK(3) intra Elp4(5)-Elp4(55)   1 4 5 752.418 3 2254.22808 -1.53414 4.20E-06 KRGEILNDR(1)-KLNIADESK(1) intra Elp1(206)-Elp1(278)   2 3 5 865.652 4 3458.56707 -2.91785 1.42E-05 GDCDYFAVSSVEEVPDEDDETKSIK(22)-GKGAR(2) intra Elp1(1217)-Elp1(1246)    5 5 748.639 4 2990.52365 -1.35468 1.63E-11 KGTIYEEEYLVQSVGR(1)-YTGKTGGTAK(4) intra Elp4(169)-Elp4(207)    4 4 710.737 3 2129.18844 -0.51733 1.10E-09 ELPGIYKGSR(7)-DLKIAWK(3) intra Elp6(230)-Elp6(269)    4 4 566.494 5 2827.43266 0.101682 3.11E-07 TGFAKDVTGSLHVCR(5)-ESTKLFYR(4) intra Elp3(56)-Elp3(423)    4 4 1005.88 3 3014.61888 -1.45756 5.20E-06 EVGIQEVHHKVQPDQVELIR(10)-YSKK(3) intra Elp3(12)-Elp3(56)    4 4 384.892 3 1151.65503 1.925928 7.99E-05 YSKK(3)-TNKK(3) intra Elp3(6)-Elp3(59)    4 4 480.629 3 1438.866 0.114535 4.33E-05 KYKLK(3)-HGKGPK(3) intra Elp1(814)-Elp1(844)    4 4 1150.32 4 4597.23915 -0.96971 2.76E-08 VDYLNLFISCLSEDDVTKTK(18)-SFGMEPAPLTEMQIYMKK(17) intra Elp1(1246)-Elp1(1316)    4 4 812.667 4 3246.62957 -3.16315 6.03E-07 KGTIYEEEYLVQSVGR(1)-EIYSISEKDR(8) intra Elp1(844)-Elp1(1308)    4 4 1206.94 3 3617.80709 -1.09541 2.29E-18 SFGMEPAPLTEMQIYMKK(17)-ANVKEIYSISEK(4) intra Elp4(143)-Elp4(211)    4 4 675.695 3 2024.05784 -1.92133 1.82E-05 ISDSSADKTR(8)-IAWKYK(4) intra Elp1(206)-Elp1(220) 2 2   4 785.12 3 2351.32845 -2.69774 1.49E-04 EALASLKASGLVGNQLR(7)-GKGAR(2) intra Elp3(79)-Elp3(401)   3 1 4 495.771 4 1979.05837 1.281621 9.40E-08 MKDLGTTCR(2)-KYLLPK(1) intra Elp4(173)-Elp4(204)    4 4 446.588 3 1336.74246 0.49112 1.16E-05 YKDLK(2)-KQMK(1) intra Elp1(846)-Elp1(1223)   2 2 4 636.999 3 1907.97748 1.487074 2.97E-05 TGGTAKTGASR(6)-KMFDPK(1) 206  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp1(1217)-Elp1(1232)   1 2 3 562.31 3 1680.90464 3.826812 1.45E-04 YTGKTGGTAK(4)-TAKNK(3) intra Elp1(206)-Elp1(311)   1 2 3 1027.03 4 4104.06979 -1.02873 2.73E-05 EGQLDSASEPVTGMEHQLSWKPQGSLIASIQR(21)-GKGAR(2) intra Elp2(460)-Elp2(553)   1 2 3 1146.34 4 4581.32529 -3.80194 1.57E-04 HLLWPEVEKLYGHGFEITCLDISPDQK(9)-FVSGGDEKILR(8) intra Elp4(204)-Elp4(375)    3 3 820.145 3 2457.41073 -0.95963 1.19E-04 SQPGKIQHGLVHILK(5)-YKDLK(2) intra Elp1(756)-Elp1(1217)   1 2 3 618.337 3 1851.98766 -0.92425 1.75E-05 YTGKTGGTAK(4)-NIMAKR(5) intra Elp2(647)-Elp2(680) 3    3 417.238 5 2081.15341 -0.27845 4.00E-05 FKNEKPHTR(2)-TVKVWR(3) intra Elp4(173)-Elp4(375)    3 3 388.563 6 2325.33547 1.297449 8.54E-05 SQPGKIQHGLVHILK(5)-KQMK(1) intra Elp1(198)-Elp1(278)    3 3 996.47 4 3981.84653 -1.50653 1.45E-13 GDCDYFAVSSVEEVPDEDDETKSIK(22)-HVTVGWGKK(8) intra Elp4(55)-Elp4(277)   3  3 1162.9 4 4647.54482 -1.39514 4.30E-11 LMPAPIASELTFIAPTQPVSTILSQIEQTIKR(31)-KLNIADESK(1) intra Elp3(84)-Elp3(401)    2 2 524.042 4 2092.14243 1.216936 1.12E-04 MKDLGTTCR(2)-YLLPKLK(5) intra Elp3(79)-Elp3(453)   1 1 2 647.084 3 1938.22812 -1.68179 1.88E-10 KDILIGLLR(1)-KYLLPK(1) intra Elp1(1223)-Elp1(1234)   1 1 2 621.35 3 1860.01771 -3.80757 5.11E-06 TGGTAKTGASR(6)-TAKNKR(5) intra Elp3(61)-Elp3(502)    2 2 733.14 4 2926.51232 -4.05528 1.08E-08 KFQHQGFGTLLMEEAER(1)-LKQQPR(2) intra Elp3(59)-Elp3(79)    2 2 363.232 4 1448.90066 0.547174 1.03E-04 KYLLPK(1)-YKLK(2) intra Elp1(967)-Elp1(967)   2  2 883.45 4 3527.78639 6.28766 7.97E-04 RKFLIDDYLGNYEK(2)-KFLIDDYLGNYEK(1) intra Elp4(143)-Elp4(218)    2 2 650.005 3 1946.99088 -1.22445 3.65E-08 ISDSSADKTR(8)-LADEKR(5) intra Elp1(846)-Elp1(1304)  2   2 835.468 3 2503.37598 -2.07292 2.25E-07 NFVEVLDLLKANVK(10)-KMFDPK(1) intra Elp4(176)-Elp4(399)    2 2 657.95 5 3280.68354 -5.10121 2.91E-04 GEMRVLKSEWAFK(7)-QMKKNLISEEESK(3) intra Elp3(88)-Elp3(401)    2 2 596.987 3 1787.93859 -0.84293 3.09E-05 MKDLGTTCR(2)-AKPVR(2) 207  Table A.3. Results from crosslinking mass-spectrometry of Elongator   Spectral Count       Inter- or Intra- Cross-linked peptides 40 uM 80 uM 160 uM 240 uM Total m/z Charge (+) Calculated Mass ppm Best E-value Peptide intra Elp3(14)-Elp3(56)   2  2 449.935 3 1346.78094 -0.71608 2.65E-05 KLAPEK(1)-YSKK(3) intra Elp3(78)-Elp3(88)    2 2 589.589 4 2354.32092 -1.75065 1.58E-09 LTDIINSIPDQYKK(13)-AKPVR(2) intra Elp3(19)-Elp3(423)   2  2 1111.6 3 3331.78878 3.206696 5.94E-05 EVGIQEVHHKVQPDQVELIR(10)-LAPEKER(5) intra Elp3(6)-Elp3(56)   1 1 2 429.247 3 1284.71901 -0.16688 2.61E-05 HGKGPK(3)-YSKK(3) intra Elp1(1270)-Elp1(1304)    2 2 960.879 3 2879.612 -0.57383 1.24E-11 NFVEVLDLLKANVK(10)-LNQTKPDAVR(5) intra Elp4(143)-Elp4(204)    2 2 628.33 3 1881.96836 0.297295 1.15E-04 ISDSSADKTR(8)-YKDLK(2)            208  Appendix B: Publications arising from graduate work  First author publications arising from Ph.D. studies Setiaputra, D., Cheng, D.T., Lu, S., Hansen, J., Dalwadi, U., Lam, C.H., To, J.L., Dong, M.Q., Yip, C.K. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement. EMBO Rep. e201642548. (2016). Kim, D.G.*, Setiaputra, D.*, Jung, T.Y., Chung, J.H., Leitner, A., Yoon, J.M., Aebersold, R., Hebert, H., Yip, C.K., Song, J.J. Molecular architecture of yeast chromatin assembly factor 1. Sci Rep. 6, 26702. (2016). Setiaputra, D., Ross, J.D., Lu, S., Cheng, D.T., Dong, M.Q., Yip, C.K. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 Acetyltransferase Complex. J Biol Chem. 290(16), 10057-70. (2015). *Co-first author publication Other publications during Ph.D. studies Bush, M., Setiaputra, D., Yip, C.K., Molday, R.S. Cog-wheel octameric structure of RS1, the discoidin domain containing retinal protein associated with X-linked retinoschisis. PLoS One. 11(1), e0147653. (2016) Solomonson, M., Setiaputra, D., Makepeace, K.A., Lameignere, E., Petrotchenko, E.V., Conrady, D.G., Bergeron, J.R., Vuckovic, M., DiMaio, F., Borchers, C.H., Yip, C.K., Strynadka N.C. Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism. Structure. 23(3), 571-83. (2015) 209  Chan, C.S., Song, X., Qazi, S.J., Setiaputra, D., Yip, C.K., Chao, T.C., Turner, R.J. Unusual pairing between assistants: interaction of the twin-arginine system-specific chaperone DmsD with the chaperonin GroEL. Biochem Biophys Res Commun. 456(4), 841-6. (2015) Chew, L.H., Setiaputra, D., Klionsky, D.J., Yip, C.K. Structural characterization of the Saccharomyces cerevisiae autophagy regulatory complex Atg17-Atg31-Atg29. Autophagy. 9(10), 1467-74. (2013).   


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