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- PDB-6ef3: Yeast 26S proteasome bound to ubiquitinated substrate (4D motor state) -

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Basic information

Entry
Database: PDB / ID: 6ef3
TitleYeast 26S proteasome bound to ubiquitinated substrate (4D motor state)
Components
  • (26S proteasome regulatory subunit ...) x 5
  • (Proteasome subunit alpha type- ...) x 6
  • (Proteasome subunit beta type- ...) x 8
  • 26S proteasome subunit RPT4Proteasome
  • Model substrate polypeptide
  • Probable proteasome subunit alpha type-7
  • Ubiquitin carboxyl-terminal hydrolase RPN11
  • Ubiquitin-60S ribosomal protein L40
KeywordsMOTOR PROTEIN / 26S Proteasome / ATPase / AAA+ / Protease / Ubiquitin
Function / homology
Function and homology information


proteasome storage granule assembly / peroxisome fission / proteasome regulatory particle assembly / nonfunctional rRNA decay / cellular protein-containing complex localization / proteasome-activating ATPase activity / nuclear proteasome complex / mitochondrial fission / proteasome regulatory particle, lid subcomplex / proteasome regulatory particle, base subcomplex ...proteasome storage granule assembly / peroxisome fission / proteasome regulatory particle assembly / nonfunctional rRNA decay / cellular protein-containing complex localization / proteasome-activating ATPase activity / nuclear proteasome complex / mitochondrial fission / proteasome regulatory particle, lid subcomplex / proteasome regulatory particle, base subcomplex / Lys63-specific deubiquitinase activity / cytosolic proteasome complex / proteasome binding / ribosomal large subunit export from nucleus / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / peptide catabolic process / endopeptidase activator activity / proteasomal ubiquitin-independent protein catabolic process / proteasome storage granule / proteasome core complex / proteasome core complex, beta-subunit complex / thiol-dependent ubiquitin-specific protease activity / proteasome endopeptidase complex / proteasome core complex, alpha-subunit complex / ubiquitin-dependent ERAD pathway / proteasome assembly / threonine-type endopeptidase activity / ubiquitinyl hydrolase 1 / TBP-class protein binding / proteasomal protein catabolic process / positive regulation of transcription elongation from RNA polymerase II promoter / nucleotide-excision repair / metallopeptidase activity / positive regulation of RNA polymerase II transcriptional preinitiation complex assembly / modification-dependent protein catabolic process / cytoplasmic translation / positive regulation of protein catabolic process / protein tag / ribosome biogenesis / ribosomal large subunit assembly / cytosolic large ribosomal subunit / negative regulation of DNA-binding transcription factor activity / proteasome-mediated ubiquitin-dependent protein catabolic process / positive regulation of DNA-binding transcription factor activity / ubiquitin-dependent protein catabolic process / structural constituent of ribosome / chromatin remodeling / endopeptidase activity / protein ubiquitination / ATPase activity / cell cycle / protein deubiquitination / mRNA binding / cell division / protein domain specific binding / endoplasmic reticulum membrane / ubiquitin protein ligase binding / mitochondrion / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Ribosomal L40e family / Ubiquitin conserved site / Proteasome beta 3 subunit / Proteasomal ATPase OB C-terminal domain / Ubiquitin-like domain superfamily / Nucleophile aminohydrolases, N-terminal / P-loop containing nucleoside triphosphate hydrolase / Rpn11/EIF3F, C-terminal / Proteasome beta subunit, C-terminal / Proteasome B-type subunit ...Ribosomal L40e family / Ubiquitin conserved site / Proteasome beta 3 subunit / Proteasomal ATPase OB C-terminal domain / Ubiquitin-like domain superfamily / Nucleophile aminohydrolases, N-terminal / P-loop containing nucleoside triphosphate hydrolase / Rpn11/EIF3F, C-terminal / Proteasome beta subunit, C-terminal / Proteasome B-type subunit / Proteasome alpha-type subunit / Ubiquitin domain / Proteasome subunit beta 4 / Proteasome subunit alpha6 / Proteasome beta-type subunit, conserved site / Cyclin-like / Cyclin, N-terminal / 26S proteasome regulatory subunit P45-like / Cyclin, C-terminal domain / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / Ribosomal protein L40e / Proteasome, subunit alpha/beta / Ubiquitin-like domain / JAB1/MPN/MOV34 metalloenzyme domain / Proteasome subunit alpha5 / Proteasome subunit alpha2 / Peptidase T1A, proteasome beta-subunit / Ribosomal protein L40e superfamily / Ubiquitin family / Maintenance of mitochondrial structure and function / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Proteasomal ATPase OB C-terminal domain / AAA+ lid domain / ATPase family associated with various cellular activities (AAA) / Proteasome subunit A N-terminal signature / Proteasome beta subunits C terminal / Proteasome subunit / AAA ATPase, AAA+ lid domain / Cyclin / Proteasome subunit beta Pre3 / Proteasome subunit alpha 1 / Proteasome subunit alpha 3 / MPN domain / Cyclin-like superfamily / 26S Proteasome non-ATPase regulatory subunit 14 / 26S proteasome regulatory subunit 10B / 26S Proteasome regulatory subunit 6B / 26S Proteasome regulatory subunit 7 / 26S Proteasome regulatory subunit 4 / Proteasome subunit beta 7 / Proteasome subunit beta 2 / Proteasome subunit beta 1 / Proteasome alpha-subunit, N-terminal domain / AAA+ ATPase domain
Proteasome subunit beta type-5 / 26S proteasome regulatory subunit 8 homolog / G2/mitotic-specific cyclin-B / Ubiquitin-60S ribosomal protein L40 / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-4 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit beta type-6 ...Proteasome subunit beta type-5 / 26S proteasome regulatory subunit 8 homolog / G2/mitotic-specific cyclin-B / Ubiquitin-60S ribosomal protein L40 / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-4 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit beta type-6 / Proteasome subunit beta type-2 / 26S proteasome subunit RPT4 / Proteasome subunit beta type-7 / Ubiquitin carboxyl-terminal hydrolase RPN11 / 26S proteasome regulatory subunit 4 homolog / Proteasome subunit alpha type-4 / Proteasome subunit alpha type-6 / Proteasome subunit beta type-1 / Proteasome subunit beta type-3 / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 6A / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 7 homolog
Biological speciesSaccharomyces cerevisiae S288c (yeast)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.17 Å
Authorsde la Pena, A.H. / Goodall, E.A. / Gates, S.N. / Lander, G.C. / Martin, A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM094497 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2-EB020402 United States
CitationJournal: Science / Year: 2018
Title: Substrate-engaged 26 proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.
Authors: Andres H de la Peña / Ellen A Goodall / Stephanie N Gates / Gabriel C Lander / Andreas Martin /
Abstract: The 26 proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the ...The 26 proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26 proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 15, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 17, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 24, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 12, 2018Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
1: Proteasome subunit beta type-1
2: Proteasome subunit beta type-2
3: Proteasome subunit beta type-3
4: Proteasome subunit beta type-4
5: Proteasome subunit beta type-5
6: Proteasome subunit beta type-6
7: Proteasome subunit beta type-7
A: Proteasome subunit alpha type-1
B: Proteasome subunit alpha type-2
C: Proteasome subunit alpha type-3
D: Proteasome subunit alpha type-4
E: Proteasome subunit alpha type-5
F: Proteasome subunit alpha type-6
G: Probable proteasome subunit alpha type-7
H: 26S proteasome regulatory subunit 7 homolog
I: 26S proteasome regulatory subunit 4 homolog
J: 26S proteasome regulatory subunit 8 homolog
K: 26S proteasome regulatory subunit 6B homolog
L: 26S proteasome subunit RPT4
M: 26S proteasome regulatory subunit 6A
n: Proteasome subunit beta type-7
r: Ubiquitin carboxyl-terminal hydrolase RPN11
s: Model substrate polypeptide
u: Ubiquitin-60S ribosomal protein L40
hetero molecules


Theoretical massNumber of molelcules
Total (without water)733,21530
Polymers730,33224
Non-polymers2,8836
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, purification of holoenzyme, microscopy, EM density
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area94290 Å2
ΔGint-439 kcal/mol
Surface area253890 Å2

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Components

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Proteasome subunit beta type- ... , 8 types, 8 molecules 1234567n

#1: Protein Proteasome subunit beta type-1 / PSMB1 / Macropain subunit PRE3 / Multicatalytic endopeptidase complex subunit PRE3 / Proteasome component ...Macropain subunit PRE3 / Multicatalytic endopeptidase complex subunit PRE3 / Proteasome component PRE3 / Proteinase YSCE subunit PRE3


Mass: 23573.604 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE3, YJL001W, J1407 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P38624, proteasome endopeptidase complex
#2: Protein Proteasome subunit beta type-2 / PSMB2 / Macropain subunit PUP1 / Multicatalytic endopeptidase complex subunit PUP1 / Proteasome component ...Macropain subunit PUP1 / Multicatalytic endopeptidase complex subunit PUP1 / Proteasome component PUP1 / Proteinase YSCE subunit PUP1


Mass: 28299.889 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PUP1, YOR157C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P25043, proteasome endopeptidase complex
#3: Protein Proteasome subunit beta type-3 / PSMB3 / Macropain subunit PUP3 / Multicatalytic endopeptidase complex subunit PUP3 / Proteasome component PUP3


Mass: 22627.842 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PUP3, YER094C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P25451, proteasome endopeptidase complex
#4: Protein Proteasome subunit beta type-4 / PSMB4 / Macropain subunit C11 / Multicatalytic endopeptidase complex subunit C11 / Proteasome component C11 ...Macropain subunit C11 / Multicatalytic endopeptidase complex subunit C11 / Proteasome component C11 / Proteinase YSCE subunit 11


Mass: 22545.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE1, YER012W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P22141, proteasome endopeptidase complex
#5: Protein Proteasome subunit beta type-5 / PSMB5 / Macropain subunit PRE2 / Multicatalytic endopeptidase complex subunit PRE2 / Proteasome component ...Macropain subunit PRE2 / Multicatalytic endopeptidase complex subunit PRE2 / Proteasome component PRE2 / Proteinase YSCE subunit PRE2


Mass: 31670.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE2, DOA3, PRG1, YPR103W, P8283.10 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P30656, proteasome endopeptidase complex
#6: Protein Proteasome subunit beta type-6 / / Multicatalytic endopeptidase complex subunit C5 / Proteasome component C5


Mass: 26905.076 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE7, PRS3, PTS1, YBL041W, YBL0407 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P23724, proteasome endopeptidase complex
#7: Protein Proteasome subunit beta type-7 / / Macropain subunit PRE4 / Multicatalytic endopeptidase complex subunit PRE4 / Proteasome component ...Macropain subunit PRE4 / Multicatalytic endopeptidase complex subunit PRE4 / Proteasome component PRE4 / Proteinase YSCE subunit PRE4


Mass: 29471.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE4, YFR050C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P30657, proteasome endopeptidase complex
#21: Protein/peptide Proteasome subunit beta type-7 /


Mass: 1789.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Gene: PSB7 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P30657*PLUS

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Proteasome subunit alpha type- ... , 6 types, 6 molecules ABCDEF

#8: Protein Proteasome subunit alpha type-1 / / Macropain subunit C7-alpha / Multicatalytic endopeptidase complex C7 / Proteasome component C7- ...Macropain subunit C7-alpha / Multicatalytic endopeptidase complex C7 / Proteasome component C7-alpha / Proteasome component Y8 / Proteinase YSCE subunit 7 / SCL1 suppressor protein


Mass: 28033.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: SCL1, PRC2, PRS2, YGL011C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P21243, proteasome endopeptidase complex
#9: Protein Proteasome subunit alpha type-2 / / Macropain subunit Y7 / Multicatalytic endopeptidase complex subunit Y7 / Proteasome component Y7 / ...Macropain subunit Y7 / Multicatalytic endopeptidase complex subunit Y7 / Proteasome component Y7 / Proteinase YSCE subunit 7


Mass: 27191.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE8, PRS4, YML092C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P23639, proteasome endopeptidase complex
#10: Protein Proteasome subunit alpha type-3 / / Macropain subunit Y13 / Multicatalytic endopeptidase complex subunit Y13 / Proteasome component Y13 ...Macropain subunit Y13 / Multicatalytic endopeptidase complex subunit Y13 / Proteasome component Y13 / Proteinase YSCE subunit 13


Mass: 28748.230 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE9, PRS5, YGR135W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P23638, proteasome endopeptidase complex
#11: Protein Proteasome subunit alpha type-4 / / Macropain subunit PRE6 / Multicatalytic endopeptidase complex subunit PRE6 / Proteasome component ...Macropain subunit PRE6 / Multicatalytic endopeptidase complex subunit PRE6 / Proteasome component PRE6 / Proteinase YSCE subunit PRE6


Mass: 28478.111 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE6, YOL038W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P40303, proteasome endopeptidase complex
#12: Protein Proteasome subunit alpha type-5 / / Macropain subunit PUP2 / Multicatalytic endopeptidase complex subunit PUP2 / Proteasome component ...Macropain subunit PUP2 / Multicatalytic endopeptidase complex subunit PUP2 / Proteasome component PUP2 / Proteinase YSCE subunit PUP2


Mass: 28649.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PUP2, DOA5, YGR253C, G9155 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P32379, proteasome endopeptidase complex
#13: Protein Proteasome subunit alpha type-6 / / Macropain subunit PRE5 / Multicatalytic endopeptidase complex subunit PRE5 / Proteasome component ...Macropain subunit PRE5 / Multicatalytic endopeptidase complex subunit PRE5 / Proteasome component PRE5 / Proteinase YSCE subunit PRE5


Mass: 25634.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE5, YMR314W, YM9924.06 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P40302, proteasome endopeptidase complex

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Protein , 4 types, 4 molecules GLru

#14: Protein Probable proteasome subunit alpha type-7 / Macropain subunit C1 / Multicatalytic endopeptidase complex subunit C1 / Proteasome component C1 / ...Macropain subunit C1 / Multicatalytic endopeptidase complex subunit C1 / Proteasome component C1 / Proteinase YSCE subunit 1


Mass: 31575.068 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: PRE10, PRC1, PRS1, YOR362C, O6650 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40
References: UniProt: P21242, proteasome endopeptidase complex
#19: Protein 26S proteasome subunit RPT4 / Proteasome / 26S protease subunit SUG2 / Proteasomal cap subunit


Mass: 49480.137 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT4, CRL13, PCS1, SUG2, YOR259C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P53549
#22: Protein Ubiquitin carboxyl-terminal hydrolase RPN11 / 26S proteasome regulatory subunit RPN11 / Protein MPR1


Mass: 34442.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPN11, MPR1, YFR004W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P43588, ubiquitinyl hydrolase 1
#24: Protein Ubiquitin-60S ribosomal protein L40


Mass: 14583.077 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPL40A, UBI1, YIL148W / Production host: Escherichia coli B (bacteria) / References: UniProt: P0CH08

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26S proteasome regulatory subunit ... , 5 types, 5 molecules HIJKM

#15: Protein 26S proteasome regulatory subunit 7 homolog / Protein CIM5 / Tat-binding homolog 3


Mass: 52054.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT1, CIM5, YTA3, YKL145W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P33299
#16: Protein 26S proteasome regulatory subunit 4 homolog / Tat-binding homolog 5


Mass: 48898.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT2, YHS4, YTA5, YDL007W, D2920 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P40327
#17: Protein 26S proteasome regulatory subunit 8 homolog / Protein CIM3 / Protein SUG1 / Tat-binding protein TBY1


Mass: 45342.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT6, CIM3, CRL3, SUG1, TBPY, TBY1, YGL048C / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: Q01939
#18: Protein 26S proteasome regulatory subunit 6B homolog / Protein YNT1 / Tat-binding homolog 2


Mass: 47953.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT3, YNT1, YTA2, YDR394W, D9509.14 / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P33298
#20: Protein 26S proteasome regulatory subunit 6A / Tat-binding protein homolog 1 / TBP-1


Mass: 48315.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Strain: ATCC 204508 / S288c / Gene: RPT5, YTA1, YOR117W, O3258, YOR3258W / Production host: Saccharomyces cerevisiae W303 (yeast) / Strain (production host): YYS40 / References: UniProt: P33297

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Protein/peptide , 1 types, 1 molecules s

#23: Protein/peptide Model substrate polypeptide


Mass: 4068.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: P07818*PLUS

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Non-polymers , 2 types, 6 molecules

#25: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#26: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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
1Substrate-engaged 26S proteasome in the 4D stateCOMPLEXYeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 240MULTIPLE SOURCES
2ProteasomeCOMPLEX1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 231RECOMBINANT
3substrateCOMPLEX241RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292ATCC 204508 / S288c
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
92Saccharomyces cerevisiae W303 (yeast)580240
103Escherichia coli BL21 (bacteria)511693BL21
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
225 mMNaClSodium chloride1
325 mMKCl1
410 mMMgCl21
51 mMTCEP1
65 mMATPAdenosine triphosphate1
70.05 % (w/v)Nonidet P-401
86 mMortho-phenanthroline1
SpecimenConc.: 25 mg/ml
Details: 26S proteasomes were diluted to a concentration of 20 micromolar in a buffer with an ATP regeneration system, and 6 mM ortho-phenanthroline. This solution was mixed with an equal volume of ...Details: 26S proteasomes were diluted to a concentration of 20 micromolar in a buffer with an ATP regeneration system, and 6 mM ortho-phenanthroline. This solution was mixed with an equal volume of 50 micromolar ubiquitinated model substrate
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K
Details: specimens were manually blotted with Whatman #1 filter paper

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: images were acquired in nanoprobe mode
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -1000 nm / Calibrated defocus min: -1500 nm / Calibrated defocus max: -3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.25 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11656
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

EM software
IDNameVersionCategory
2Leginon3.2image acquisition
4MotionCorr22CTF correction
5RELION2.1CTF correction
6CTFFIND4CTF correction
7GctfCTF correction
10UCSF Chimera1.12model fitting
11PHENIX1.11-2580model fitting
13RELION2.1initial Euler assignment
14RELION2.1final Euler assignment
16RELION2.13D reconstruction
17PHENIX1.11-2580model refinement
Image processingDetails: Camera was operated in counting mode
CTF correctionDetails: CTF correction was performed by Relion during reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 579361
Details: Particles were selected using the Relion template-based particle picker
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48335 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5MPC

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