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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


peroxisome fission / proteasome storage granule assembly / proteasome regulatory particle assembly / nonfunctional rRNA decay / mitotic cell cycle phase transition / cytosolic proteasome complex / proteasome regulatory particle, lid subcomplex / proteasome-activating activity / protein-containing complex localization / mitochondrial fission ...peroxisome fission / proteasome storage granule assembly / proteasome regulatory particle assembly / nonfunctional rRNA decay / mitotic cell cycle phase transition / cytosolic proteasome complex / proteasome regulatory particle, lid subcomplex / proteasome-activating activity / protein-containing complex localization / mitochondrial fission / proteasome regulatory particle, base subcomplex / cyclin-dependent protein serine/threonine kinase regulator activity / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Cross-presentation of soluble exogenous antigens (endosomes) / TNFR2 non-canonical NF-kB pathway / proteasomal ubiquitin-independent protein catabolic process / Ub-specific processing proteases / peptide catabolic process / proteasome binding / protein deubiquitination / proteasome storage granule / endopeptidase activator activity / ribosomal large subunit export from nucleus / proteasome assembly / proteasome endopeptidase complex / positive regulation of RNA polymerase II transcription preinitiation complex assembly / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / : / Neutrophil degranulation / proteasome complex / proteasomal protein catabolic process / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / modification-dependent protein catabolic process / protein tag activity / positive regulation of protein catabolic process / ribosomal large subunit assembly / metallopeptidase activity / ribosome biogenesis / cytoplasmic translation / cytosolic large ribosomal subunit / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / endopeptidase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / protein ubiquitination / structural constituent of ribosome / chromatin remodeling / cell division / protein domain specific binding / mRNA binding / ubiquitin protein ligase binding / endoplasmic reticulum membrane / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / : / 26S proteasome regulatory subunit 7, OB domain / Cyclin, C-terminal domain / Cyclin_C / Maintenance of mitochondrial structure and function ...: / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / : / 26S proteasome regulatory subunit 7, OB domain / Cyclin, C-terminal domain / Cyclin_C / Maintenance of mitochondrial structure and function / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / Cyclin, N-terminal / Cyclin, N-terminal domain / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / Proteasome subunit / Proteasome, subunit alpha/beta / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / Nucleophile aminohydrolases, N-terminal / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / G2/mitotic-specific cyclin-B / Ubiquitin-ribosomal protein eL40A fusion protein / 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 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / G2/mitotic-specific cyclin-B / Ubiquitin-ribosomal protein eL40A fusion protein / 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 / Proteasome subunit beta type-3 / Proteasome subunit beta type-5 / Proteasome subunit beta type-7 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit beta type-1 / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / 26S proteasome regulatory subunit 4 homolog / Ubiquitin carboxyl-terminal hydrolase RPN11 / 26S proteasome subunit RPT4 / 26S proteasome regulatory subunit 8 homolog
Similarity search - Component
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.
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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  • Deposited structure unit
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Structure viewerMolecule:
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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
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 ATP#1-#240MULTIPLE SOURCES
2ProteasomeCOMPLEX#1-#231RECOMBINANT
3substrateCOMPLEX#241RECOMBINANT
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 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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
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 / Specimen holder 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

5mpc

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