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- PDB-7ktr: Cryo-EM structure of the human SAGA coactivator complex (TRRAP, core) -

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Basic information

Entry
Database: PDB / ID: 7ktr
TitleCryo-EM structure of the human SAGA coactivator complex (TRRAP, core)
Components
  • (Transcription initiation factor TFIID subunit ...) x 3
  • Ataxin-7Ataxin 7
  • Isoform 3 of Transcription factor SPT20 homolog
  • STAGA complex 65 subunit gamma,DhaA,STAGA complex 65 subunit gamma,STAGA complex 65 subunit gamma,DhaA,STAGA complex 65 subunit gamma,STAGA complex 65 subunit gamma,DhaA
  • TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L
  • TAF6-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 6L
  • Transcription initiation protein SPT3 homolog,Transcription initiation protein SPT3 homolog,Transcription initiation protein SPT3 homologTranscription (biology)
  • Transcriptional adapter 1
  • Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein
KeywordsTRANSCRIPTION / coactivator / gene regulation / chromatin
Function / homology
Function and homology information


regulation of nucleobase-containing compound metabolic process / regulation of macromolecule metabolic process / SAGA-type complex / positive regulation of response to stimulus / : / regulation of somatic stem cell population maintenance / SAGA complex assembly / lateral mesodermal cell differentiation / allantois development / regulation of cellular response to stress ...regulation of nucleobase-containing compound metabolic process / regulation of macromolecule metabolic process / SAGA-type complex / positive regulation of response to stimulus / : / regulation of somatic stem cell population maintenance / SAGA complex assembly / lateral mesodermal cell differentiation / allantois development / regulation of cellular response to stress / transcription factor TFTC complex / SLIK (SAGA-like) complex / negative regulation of microtubule depolymerization / hepatocyte differentiation / maintenance of protein location in nucleus / SAGA complex / RNA polymerase binding / limb development / transcription preinitiation complex / nucleus organization / negative regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / RNA polymerase II general transcription initiation factor activity / transcription factor TFIID complex / regulation of RNA splicing / HIV Transcription Initiation / RNA Polymerase II HIV Promoter Escape / Transcription of the HIV genome / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / histone deacetylase complex / histone acetyltransferase complex / gastrulation / positive regulation of transcription initiation by RNA polymerase II / embryonic placenta development / regulation of DNA repair / somitogenesis / RNA polymerase II preinitiation complex assembly / RNA Polymerase II Pre-transcription Events / TBP-class protein binding / visual perception / male germ cell nucleus / nuclear estrogen receptor binding / promoter-specific chromatin binding / transcription initiation at RNA polymerase II promoter / DNA-templated transcription initiation / transcription coregulator activity / G1/S transition of mitotic cell cycle / multicellular organism growth / mRNA transcription by RNA polymerase II / cytoplasmic ribonucleoprotein granule / microtubule cytoskeleton organization / nuclear matrix / autophagy / transcription corepressor activity / microtubule cytoskeleton / kinase activity / HATs acetylate histones / positive regulation of cell growth / DNA-binding transcription factor binding / Regulation of TP53 Activity through Phosphorylation / transcription by RNA polymerase II / transcription coactivator activity / protein stabilization / Ub-specific processing proteases / nuclear speck / chromatin remodeling / protein heterodimerization activity / phosphorylation / focal adhesion / apoptotic process / nucleolus / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / perinuclear region of cytoplasm / positive regulation of DNA-templated transcription / Golgi apparatus / negative regulation of transcription by RNA polymerase II / enzyme binding / positive regulation of transcription by RNA polymerase II / DNA binding / extracellular exosome / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
STAGA complex 65 subunit gamma / Transcription-associated protein 1 / Transcription factor Spt20 / Transcriptional coactivator Hfi1/Transcriptional adapter 1 / Transcriptional regulator of RNA polII, SAGA, subunit / Spt20-like, SEP domain / Spt20, SEP domain / SCA7 domain / SCA7, zinc-binding domain / SCA7 domain profile. ...STAGA complex 65 subunit gamma / Transcription-associated protein 1 / Transcription factor Spt20 / Transcriptional coactivator Hfi1/Transcriptional adapter 1 / Transcriptional regulator of RNA polII, SAGA, subunit / Spt20-like, SEP domain / Spt20, SEP domain / SCA7 domain / SCA7, zinc-binding domain / SCA7 domain profile. / Transcription initiation factor IID, subunit 13 / Transcription initiation factor IID, 18kD subunit / Bromodomain associated / Bromodomain transcription factors and PHD domain containing proteins / Bromodomain associated domain / Transcription initiation factor IID, 31kD subunit / Transcription initiation factor TFIID subunit 12 / Transcription initiation factor TFIID subunit A / TFIID subunit TAF5, NTD2 domain superfamily / WD40 associated region in TFIID subunit, NTD2 domain / Transcription initiation factor TAFII31 / TAF6, C-terminal HEAT repeat domain superfamily / TATA box binding protein associated factor (TAF) / Transcription initiation factor TFIID subunit 12 domain / TFIID subunit TAF5, NTD2 domain / Transcription initiation factor TFIID subunit 6 / TAF6 C-terminal HEAT repeat domain / TAF6, C-terminal HEAT repeat domain / Transcription initiation factor TFIID 23-30kDa subunit / Transcription initiation factor TFIID, 23-30kDa subunit / PIK-related kinase, FAT / FAT domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone-fold / Armadillo-like helical / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
INOSITOL HEXAKISPHOSPHATE / Transformation/transcription domain-associated protein / Ataxin-7 / Transcription initiation protein SPT3 homolog / TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L / STAGA complex 65 subunit gamma / Transcription initiation factor TFIID subunit 10 / Transcription initiation factor TFIID subunit 12 / Transcription factor SPT20 homolog / Transcriptional adapter 1 ...INOSITOL HEXAKISPHOSPHATE / Transformation/transcription domain-associated protein / Ataxin-7 / Transcription initiation protein SPT3 homolog / TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L / STAGA complex 65 subunit gamma / Transcription initiation factor TFIID subunit 10 / Transcription initiation factor TFIID subunit 12 / Transcription factor SPT20 homolog / Transcriptional adapter 1 / Transcription initiation factor TFIID subunit 9B / TAF6-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 6L
Similarity search - Component
Biological speciesHomo sapiens (human)
unclassified Rhodococcus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsHerbst, D.A. / Esbin, M.N. / Nogales, E.
Funding support United States, European Union, Switzerland, 6items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)CC30250 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM127018 United States
European Molecular Biology Organization (EMBO)ALTF 1002-2018European Union
Swiss National Science FoundationP2BSP3_181878 Switzerland
Howard Hughes Medical Institute (HHMI)CC34430 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM098218 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structure of the human SAGA coactivator complex.
Authors: Dominik A Herbst / Meagan N Esbin / Robert K Louder / Claire Dugast-Darzacq / Gina M Dailey / Qianglin Fang / Xavier Darzacq / Robert Tjian / Eva Nogales /
Abstract: The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are ...The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.
History
DepositionNov 24, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 10, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ASTM ..._citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year / _citation_author.name
Revision 1.2Dec 8, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Dec 22, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-23027
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein
B: TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L
C: Isoform 3 of Transcription factor SPT20 homolog
D: STAGA complex 65 subunit gamma,DhaA,STAGA complex 65 subunit gamma,STAGA complex 65 subunit gamma,DhaA,STAGA complex 65 subunit gamma,STAGA complex 65 subunit gamma,DhaA
E: Transcription initiation factor TFIID subunit 9B
F: TAF6-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 6L
G: Transcription initiation factor TFIID subunit 12
H: Transcription initiation factor TFIID subunit 10
I: Transcription initiation protein SPT3 homolog,Transcription initiation protein SPT3 homolog,Transcription initiation protein SPT3 homolog
J: Transcriptional adapter 1
N: Ataxin-7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)887,72812
Polymers887,06811
Non-polymers6601
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area82400 Å2
ΔGint-510 kcal/mol
Surface area236220 Å2

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Components

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Protein , 8 types, 8 molecules ABCDFIJN

#1: Protein Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein,Transformation/transcription domain-associated protein / TRRAP


Mass: 353170.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HeLa / References: UniProt: F2Z2U4
#2: Protein TAF5-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 5L / TAF5L / PCAF-associated factor 65 beta / PAF65-beta


Mass: 66223.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O75529
#3: Protein Isoform 3 of Transcription factor SPT20 homolog / p38-interacting protein / p38IP / SUPT20H


Mass: 88129.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q8NEM7
#4: Protein STAGA complex 65 subunit gamma,DhaA,STAGA complex 65 subunit gamma,STAGA complex 65 subunit gamma,DhaA,STAGA complex 65 subunit gamma,STAGA complex 65 subunit gamma,DhaA / Adenocarcinoma antigen ART1 / SPTF-associated factor 65 gamma / STAF65gamma / Suppressor of Ty 7- ...Adenocarcinoma antigen ART1 / SPTF-associated factor 65 gamma / STAF65gamma / Suppressor of Ty 7-like / SUPT7L / Adenocarcinoma antigen ART1 / SPTF-associated factor 65 gamma / STAF65gamma / Suppressor of Ty 7-like


Mass: 78079.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) unclassified Rhodococcus (bacteria)
Gene: SUPT7L, KIAA0764 / Production host: Homo sapiens (human) / References: UniProt: O94864
#6: Protein TAF6-like RNA polymerase II p300/CBP-associated factor-associated factor 65 kDa subunit 6L / TAF6L / PCAF-associated factor 65-alpha / PAF65-alpha


Mass: 67903.289 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9Y6J9
#9: Protein Transcription initiation protein SPT3 homolog,Transcription initiation protein SPT3 homolog,Transcription initiation protein SPT3 homolog / Transcription (biology) / SPT3-like protein / SUPT3H / SPT3-like protein


Mass: 33197.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O75486
#10: Protein Transcriptional adapter 1 / SPT3-associated factor 42 / STAF42 / Transcriptional adapter 1-like protein


Mass: 37432.531 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q96BN2
#11: Protein Ataxin-7 / Ataxin 7 / Spinocerebellar ataxia type 7 protein


Mass: 95597.742 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O15265

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Transcription initiation factor TFIID subunit ... , 3 types, 3 molecules EGH

#5: Protein Transcription initiation factor TFIID subunit 9B / Neuronal cell death-related protein 7 / DN-7 / Transcription initiation factor TFIID subunit 9-like ...Neuronal cell death-related protein 7 / DN-7 / Transcription initiation factor TFIID subunit 9-like / Transcription-associated factor TAFII31L / TAF9B


Mass: 27654.861 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9HBM6
#7: Protein Transcription initiation factor TFIID subunit 12 / Transcription initiation factor TFIID 20/15 kDa subunits / TAFII20/TAFII15


Mass: 17948.467 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q16514
#8: Protein Transcription initiation factor TFIID subunit 10 / STAF28 / Transcription initiation factor TFIID 30 kDa subunit / TAFII30


Mass: 21731.248 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q12962

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Non-polymers , 1 types, 1 molecules

#12: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / Phytic acid


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Sequence detailsPortions of chains A, D and I were not fully discernible in the map, and so many of the residues ...Portions of chains A, D and I were not fully discernible in the map, and so many of the residues were modeled as unknown (UNK) due to not knowing the register in these regions. The full sequence of the chains are as follows. Chain A: MAFVATQGATVVDQTTLMKKYLQFVAALTDVNTPDETKLKMMQEVSENFENVTSSPQYST FLEHIIPRFLTFLQDGEVQFLQEKPAQQLRKLVLEIIHRIPTNEHLRPHTKNVLSVMFRF LETENEENVLICLRIIIELHKQFRPPITQEIHHFLDFVKQIYKELPKVVNRYFENPQVIP ENTVPPPEMVGMITTIAVKVNPEREDSETRTHSIIPRGSLSLKVLAELPIIVVLMYQLYK LNIHNVVAEFVPLIMNTIAIQVSAQARQHKLYNKELYADFIAAQIKTLSFLAYIIRIYQE LVTKYSQQMVKGMLQLLSNCPAETAHLRKELLIAAKHILTTELRNQFIPCMDKLFDESIL IGSGYTARETLRPLAYSTLADLVHHVRQHLPLSDLSLAVQLFAKNIDDESLPSSIQTMSC KLLLNLVDCIRSKSEQESGNGRDVLMRMLEVFVLKFHTIARYQLSAIFKKCKPQSELGAV EAALPGVPTAPAAPGPAPSPAPVPAPPPPPPPPPPATPVTPAPVPPFEKQGEKDKEDKQT FQVTDCRSLVKTLVCGVKTITWGITSCKAPGAQFIPNKQLQPKETQIYIKLVKYAMQALD IYQVQIAGNGQTYIRVANCQTVRMKEEKEVLEHFAGVFTMMNPLTFKEIFQTTVPYMVER ISKNYALQIVANSFLANPTTSALFATILVEYLLDRLPEMGSNVELSNLYLKLFKLVFGSV SLFAAENEQMLKPHLHKIVNSSMELAQTAKEPYNYFLLLRALFRSIGGGSHDLLYQEFLP LLPNLLQGLNMLQSGLHKQHMKDLFVELCLTVPVRLSSLLPYLPMLMDPLVSALNGSQTL VSQGLRTLELCVDNLQPDFLYDHIQPVRAELMQALWRTLRNPADSISHVAYRVLGKFGGS NRKMLKESQKLHYVVTEVQGPSITVEFSDCKASLQLPMEKAIETALDCLKSANTEPYYRR QAWEVIKCFLVAMMSLEDNKHALYQLLAHPNFTEKTIPNVIISHRYKAQDTPARKTFEQA LTGAFMSAVIKDLRPSALPFVASLIRHYTMVAVAQQCGPFLLPCYQVGSQPSTAMFHSEE NGSKGMDPLVLIDAIAICMAYEEKELCKIGEVALAVIFDVASIILGSKERACQLPLFSYI VERLCACCYEQAWYAKLGGVVSIKFLMERLPLTWVLQNQQTFLKALLFVMMDLTGEVSNG AVAMAKTTLEQLLMRCATPLKDEERAEEIVAAQEKSFHHVTHDLVREVTSPNSTVRKQAM HSLQVLAQVTGKSVTVIMEPHKEVLQDMVPPKKHLLRHQPANAQIGLMEGNTFCTTLQPR LFTMDLNVVEHKVFYTELLNLCEAEDSALTKLPCYKSLPSLVPLRIAALNALAACNYLPQ SREKIIAALFKALNSTNSELQEAGEACMRKFLEGATIEVDQIHTHMRPLLMMLGDYRSLT LNVVNRLTSVTRLFPNSFNDKFCDQMMQHLRKWMEVVVITHKGGQRSDGNEMKICSAIIN LFHLIPAAPQTLVKPLLEVVMKTERAMLIEAGSPFREPLIKFLTRHPSQTVELFMMEATL NDPQWSRMFMSFLKHKDARPLRDVLAANPNRFITLLLPGGAQTAVRPGSPSTSTMRLDLQ FQAIKIISIIVKNDDSWLASQHSLVSQLRRVWVSENFQERHRKENMAATNWKEPKLLAYC LLNYCKRNYGDIELLFQLLRAFTGRFLCNMTFLKEYMEEEIPKNYSIAQKRALFFRFVDF NDPNFGDELKAKVLQHILNPAFLYSFEKGEGEQLLGPPNPEGDNPESITSVFITKVLDPE KQADMLDSLRIYLLQYATLLVEHAPHHIHDNNKNRNSKLRRLMTFAWPCLLSKACVDPAC KYSGHLLLAHIIAKFAIHKKIVLQVFHSLLKAHAMEARAIVRQAMAILTPAVPARMEDGH QMLTHWTRKIIVEEGHTVPQLVHILHLIVQHFKVYYPVRHHLVQHMVSAMQRLGFTPSVT IEQRRLAVDLSEVVIKWELQRIKDQQPDSDMDPNSSGEGVNSVSSSIKRGLSVDSAQEVK RFRTATGAISAVFGRSQSLPGADSLLAKPIDKQHTDTVVNFLIRVACQVNDNTNTAGSPG EVLSRRCVNLLKTALRPDMWPKSELKLQWFDKLLMTVEQPNQVNYGNICTGLEVLSFLLT VLQSPAILSSFKPLQRGIAACMTCGNTKVLRAVHSLLSRLMSIFPTEPSTSSVASKYEEL ECLYAAVGKVIYEGLTNYEKATNANPSQLFGTLMILKSACSNNPSYIDRLISVFMRSLQK MVREHLNPQAASGSTEATSAGTSELVMLSLELVKTRLAVMSMEMRKNFIQAILTSLIEKS PDAKILRAVVKIVEEWVKNNSPMAANQTPTLREKSILLVKMMTYIEKRFPEDLELNAQFL DLVNYVYRDETLSGSELTAKLEPAFLSGLRCAQPLIRAKFFEVFDNSMKRRVYERLLYVT CSQNWEAMGNHFWIKQCIELLLAVCEKSTPIGTSCQGAMLPSITNVINLADSHDRAAFAM VTHVKQEPRERENSESKEEDVEIDIELAPGDQTSTPKTKELSEKDIGNQLHMLTNRHDKF LDTLREVKTGALLSAFVQLCHISTTLAEKTWVQLFPRLWKILSDRQQHALAGEISPFLCS GSHQVQRDCQPSALNCFVEAMSQCVPPIPIRPCVLKYLGKTHNLWFRSTLMLEHQAFEKG LSLQIKPKQTTEFYEQESITPPQQEILDSLAELYSLLQEEDMWAGLWQKRCKYSETATAI AYEQHGFFEQAQESYEKAMDKAKKEHERSNASPAIFPEYQLWEDHWIRCSKELNQWEALT EYGQSKGHINPYLVLECAWRVSNWTAMKEALVQVEVSCPKEMAWKVNMYRGYLAICHPEE QQLSFIERLVEMASSLAIREWRRLPHVVSHVHTPLLQAAQQIIELQEAAQINAGLQPTNL GRNNSLHDMKTVVKTWRNRLPIVSDDLSHWSSIFMWRQHHYQAIVTAYENSSQHDPSSNN AMLGVHASASAIIQYGKIARKQGLVNVALDILSRIHTIPTVPIVDCFQKIRQQVKCYLQL AGVMGKNECMQGLEVIESTNLKYFTKEMTAEFYALKGMFLAQINKSEEANKAFSAAVQMH DVLVKAWAMWGDYLENIFVKERQLHLGVSAITCYLHACRHQNESKSRKYLAKVLWLLSFD DDKNTLADAVDKYCIGVPPIQWLAWIPQLLTCLVGSEGKLLLNLISQVGRVYPQAVYFPI RTLYLTLKIEQRERYKSDSGQQQPSSVGNQSHSASDPGPIRATAPMWRCSRIMHMQRELH PTLLSSLEGIVDQMVWFRENWHEEVLRQLQQGLAKCYSVAFEKSGAVSDAKITPHTLNFV KKLVSTFGVGLENVSNVSTMFSSAASESLARRAQATAQDPVFQKLKGQFTTDFDFSVPGS MKLHNLISKLKKWIKILEAKTKQLPKFFLIEEKCRFLSNFSAQTAEVEIPGEFLMPKPTH YYIKIARFMPRVEIVQKHNTAARRLYIRGHNGKIYPYLVMNDACLTESRREERVLQLLRL LNPCLEKRKETTKRHLFFTVPRVVAVSPQMRLVEDNPSSLSLVEIYKQRCAKKGIEHDNP ISRYYDRLATVQARGTQASHQVLRDILKEVQSNMVPRSMLKEWALHTFPNATDYWTFRKM FTIQLALIGFAEFVLHLNRLNPEMLQIAQDTGKLNVAYFRFDINDATGDLDANRPVPFRL TPNISEFLTTIGVSGPLTASMIAVARCFAQPNFKVDGILKTVLRDEIIAWHKKTQEDTSS PLSAAGQPENMDSQQLVSLVQKAVTAIMTRLHNLAQFEGGESKVNTLVAAANSLDNLCRM DPAWHPWL Chain D: MNLQRYWGEIPISSSQTNRSSFDLLPREFRLVEVHDPPLHQPSANKPKPPTMLDIPSEPC SLTIHTIQLIQHNRRLRNLIATAQAQNQQQTEGVKTEESEPLPSCPGSPPLPDDLLPLDC KNPNAPFQIRHSDPESDFYRGKGEPVTELSWHSCRQLLYQAVATILAHAGFDCANESVLE TLTDVAHEYCLKFTKLLRFAVDREARLGQTPFPDVMEQVFHEVGIGSVLSLQKFWQHRIK DYHSYMLQISKQLSEEYERIVNPEKATEDAKPVKIKEEPVSDITFPVSEELEADLASGDQ SLPMGVLGAQSERFPSNLEVEASPQASSAEVNASPLWNLAHVKMEPQESEEGNVSGHGVL GSDVFEEPMSGMSEAGIPQSPDDSDSSYGSHSTDSLMGSSPVFNQRCKKRMRKIGTSGED LYFQSGGSMAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNII PHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALG FHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGT LPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQS PVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLST LEISGDYKDHDGDYKDHDIDYKDDDDKGS Chain I: MNNTAASPMSTATSSSGRSTGKSISFATELQSMMYSLGDARRPLHETAVLVEDVVHTQLI NLLQQAAEVSQLRGARVITPEDLLFLMRKDKKKLRRLLKYMFIRDYKSKIVKGIDEDDLL EDKLSGSNNANKRQKIAQDFLNSIDQTGELLAMFEDDEIDEVKQERMERAERQTRIMDSA QYAEFCESRQLSFSKKASKFRDWLDCSSMEIKPNVVAMEILAYLAYETVAQLVDLALLVR QDMVTKAGDPFSHAISATFIQYHNSAESTAACGVEAHSDAIQPCHIREAIRRYSHRIGPL SPFTNAYRRNGMAFLAC

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1human SAGACOMPLEXendogeneous human SAGA purified from HeLa cells#1-#110MULTIPLE SOURCES
2human SAGACOMPLEXendogeneous human SAGA purified from HeLa cells#1-#3, #5-#111NATURAL
3STAGA complex 65 subunit gammaCOMPLEX#41RECOMBINANT
Molecular weightValue: 1.420272 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrainTissue
22Homo sapiens (human)9606HeLacervix
33Homo sapiens (human)9606
43unclassified Rhodococcus (bacteria)192944
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.8
Details: Buffers were freshly prepared and 0.22 um filtered.
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid1
2200 mMsodium chlorideNaClSodium chloride1
30.2 mMEthylenediaminetetraacetic acid1
40.5 mMtris(2-carboxyethyl)phosphine1
50.01 % (v/v)nonyl phenoxypolyethoxylethanol1
62.5 % (v/v)Glycerol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was crosslinked with 1 mM bis(sulfosuccinimidyl)suberate (BS3) prior to freezing
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: blot force 0, blot time 3 sec

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Nominal defocus max: 3400 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10224
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.7.0CTF correction
7Coot0.8.9.2model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2438694
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209435 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementDetails: Residues for which the register could not be unambiguously assigned were modeled as UNKs. Additional density corresponding to the linkers I259-I277 and J232-J247 can be observed at the ...Details: Residues for which the register could not be unambiguously assigned were modeled as UNKs. Additional density corresponding to the linkers I259-I277 and J232-J247 can be observed at the interface between the core and TRRAP module, but could not be clearly assigned to one of the subunits. Connected loop density is observed but not modeled due to insufficient information on register, linker length, or confidence in backbone assignment: A1689-A1692, A1722-A1728, A1769-A1786, A1800-1805, A1823-A1837, A2181-A2192, A2209-A2217, A2239-A2248, A2291-A2299, C27-C30 Density corresponding to H182-H187 is observed, might be explained by multiple main chain conformations and was not modeled. Continuous density for linkers connecting an unassigned (UNK) with an assigned region, corresponding to D48-D51 and D118, is observed but was omitted, because the register in the unassigned (UNK) regions could not be assigned. Region I174-I185 and its connection is observed in the LocSpiral (Kaur, S. et al., bioRxiv 2020) filtered multibody maps. The cis-peptide bond of A3698 is defined by coulomb density. Mass spectrometry did not distinguish between the isoforms (uniprot) Q8NEM7-2 and Q8NEM7-3 of SUPT20H (chain C). Both isoforms distinguish primarily in disordered regions. In an ordered region, Q8NEM7-2 and Q8NEM7-3 distinguish by an insertion of one amino acid (Gln56 in Q8NEM7-2), which is better explained by Q8NEM7-3. Mass spectrometry did not distinguish between the isoforms (uniprot) O94864 and O94864-2 (chain D). Both isoforms distinguish by two N-terminal amino acids in a disordered region. The sequence numbering corresponds to O94864. Mass spectrometry indicated that (uniprot) Q9HBM6 (TAF9B) and Q16594 (TAF9) were present in the sample. TAF9B explains the density slightly better (chain E), however, it is possible that both subunits are present in the complex. Mass spectrometry did not distinguish between the isoforms (uniprot) Q16514 and Q16514-2 (chain G). Both isoforms distinguish in the disordered N-terminus. Due to a 5x higher abundance of Q16514 in all cell types and the requirement of the longer N-terminal domain for transcriptional activation (Hamard, P.-J., et al. Oncogene. 2005 May 12;24(21):3472-83.), Q16514 was modeled. Mass spectrometry indicated that (uniprot) O15265 (ATXN7), Q9ULK2 (ATXN7L1), and Q5T6C5 (ATXN7L2) were present in the sample. The density is explained best by ATXN7 and ATXN7L1 (in this order) (chain N), however, it is possible that a mixture of all subunits is present. The modeled isoforms of all other subunits were determined either by mass spectrometry or density fit. The ligand INOSITOL HEXAKISPHOSPHATE (IHP) was identified by density fit and homology to mTORC2 (PDB 6ZWO, Scaliola A., et al. Sci Adv. 2020 Nov 6;6(45)). Additional density close to P4 might be explained by an alternative conformation or an ordered water molecule. Only the conformation with the better geometry was modeled.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00441124
ELECTRON MICROSCOPYf_angle_d0.53755737
ELECTRON MICROSCOPYf_dihedral_angle_d10.1475569
ELECTRON MICROSCOPYf_chiral_restr0.0366361
ELECTRON MICROSCOPYf_plane_restr0.0047167

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