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- PDB-6ef2: Yeast 26S proteasome bound to ubiquitinated substrate (5T motor state) -

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Entry
Database: PDB / ID: 6ef2
TitleYeast 26S proteasome bound to ubiquitinated substrate (5T motor state)
Components
  • (26S proteasome regulatory subunit ...) x 5
  • (Proteasome subunit alpha type- ...) x 6
  • 26S proteasome subunit RPT4
  • Probable proteasome subunit alpha type-7
  • model substrate polypeptide
KeywordsMOTOR PROTEIN / 26S Proteasome / ATPase / AAA+ / Protease / Motor protein / Ubiquitin
Function / homologyAAA+ lid domain / Proteasome subunit alpha 1 / Proteasome subunit alpha 7-like / Proteasome subunit alpha 3 / 26S proteasome regulatory subunit 8 / 26S Proteasome regulatory subunit 6B / 26S Proteasome regulatory subunit 6A / 26S Proteasome regulatory subunit 7 / 26S Proteasome regulatory subunit 4 / Proteasome subunit alpha4 ...AAA+ lid domain / Proteasome subunit alpha 1 / Proteasome subunit alpha 7-like / Proteasome subunit alpha 3 / 26S proteasome regulatory subunit 8 / 26S Proteasome regulatory subunit 6B / 26S Proteasome regulatory subunit 6A / 26S Proteasome regulatory subunit 7 / 26S Proteasome regulatory subunit 4 / Proteasome subunit alpha4 / Proteasome subunit / Proteasome subunit alpha2 / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Nucleophile aminohydrolases, N-terminal / P-loop containing nucleoside triphosphate hydrolase / Proteasome alpha-type subunit / Proteasome beta-type subunit, conserved site / ATPase family associated with various cellular activities (AAA) / Proteasome subunit A N-terminal signature / ATPase, AAA-type, conserved site / MAPK6/MAPK4 signaling / Antigen processing: Ubiquitination & Proteasome degradation / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / CDK-mediated phosphorylation and removal of Cdc6 / Orc1 removal from chromatin / Neutrophil degranulation / Ub-specific processing proteases / TNFR2 non-canonical NF-kB pathway / Proteasomal ATPase OB C-terminal domain / AUF1 (hnRNP D0) binds and destabilizes mRNA / ABC-family proteins mediated transport / SCF(Skp2)-mediated degradation of p27/p21 / SCF-beta-TrCP mediated degradation of Emi1 / Cross-presentation of soluble exogenous antigens (endosomes) / Proteasome alpha-type subunit profile. / AAA-protein family signature. / Proteasome alpha-type subunits signature. / 26S proteasome regulatory subunit P45-like / Proteasomal ATPase OB C-terminal domain / ATPase, AAA-type, core / AAA+ ATPase domain / Proteasome, subunit alpha/beta / Proteasome alpha-subunit, N-terminal domain / proteasome regulatory particle assembly / nonfunctional rRNA decay / nuclear proteasome complex / proteasome-activating ATPase activity / proteasome regulatory particle, base subcomplex / cellular protein-containing complex localization / cytosolic proteasome complex / peptide catabolic process / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / proteasome storage granule / proteasome core complex / proteasomal ubiquitin-independent protein catabolic process / proteasome endopeptidase complex / ubiquitin-dependent ERAD pathway / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / TBP-class protein binding / positive regulation of transcription elongation from RNA polymerase II promoter / proteasomal protein catabolic process / nucleotide-excision repair / positive regulation of RNA polymerase II transcriptional preinitiation complex assembly / positive regulation of protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of DNA-binding transcription factor activity / ubiquitin-dependent protein catabolic process / chromatin remodeling / positive regulation of DNA-binding transcription factor activity / endopeptidase activity / ATPase activity / mRNA binding / protein domain specific binding / ubiquitin protein ligase binding / mitochondrion / ATP binding / identical protein binding / nucleus / cytoplasm / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / 26S proteasome regulatory subunit 4 homolog / 26S proteasome subunit RPT4 / 26S proteasome regulatory subunit 8 homolog
Function and homology information
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.27 Å resolution
Authorsde la Pena, A.H. / Goodall, E.A. / Gates, S.N. / Lander, G.C. / Martin, A.
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
DateDeposition: Aug 15, 2018 / Release: Oct 17, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 17, 2018Structure modelrepositoryInitial release
1.1Oct 24, 2018Structure modelData collection / Database referencescitation / citation_author_citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
1.2Dec 12, 2018Structure modelData collection / Database referencescitation_citation.journal_volume

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Assembly

Deposited unit
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
s: model substrate polypeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)368,20320
Polyers365,32014
Non-polymers2,8836
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, purification of holoenzyme, microscopy, EM density
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)48250
ΔGint (kcal/M)-226
Surface area (Å2)142390

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Components

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

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


Mass: 26759.469 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SCL1, PRC2, PRS2, YGL011C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40
References: UniProt: P21243, proteasome endopeptidase complex
#2: Protein/peptide Proteasome subunit alpha type-2 / / Macropain subunit Y7 / Multicatalytic endopeptidase complex subunit Y7 / Proteasome component Y7 / Proteinase YSCE subunit 7


Mass: 27078.670 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PRE8, PRS4, YML092C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40
References: UniProt: P23639, proteasome endopeptidase complex
#3: Protein/peptide Proteasome subunit alpha type-3 / / Macropain subunit Y13 / Multicatalytic endopeptidase complex subunit Y13 / Proteasome component Y13 / Proteinase YSCE subunit 13


Mass: 26805.178 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PRE9, PRS5, YGR135W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40
References: UniProt: P23638, proteasome endopeptidase complex
#4: Protein/peptide Proteasome subunit alpha type-4 / / Macropain subunit PRE6 / Multicatalytic endopeptidase complex subunit PRE6 / Proteasome component PRE6 / Proteinase YSCE subunit PRE6


Mass: 26862.277 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PRE6, YOL038W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40
References: UniProt: P40303, proteasome endopeptidase complex
#5: Protein/peptide Proteasome subunit alpha type-5 / / Macropain subunit PUP2 / Multicatalytic endopeptidase complex subunit PUP2 / Proteasome component PUP2 / Proteinase YSCE subunit PUP2


Mass: 27242.596 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PUP2, DOA5, YGR253C, G9155 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40
References: UniProt: P32379, proteasome endopeptidase complex
#6: Protein/peptide Proteasome subunit alpha type-6 / / Macropain subunit PRE5 / Multicatalytic endopeptidase complex subunit PRE5 / Proteasome component PRE5 / Proteinase YSCE subunit PRE5


Mass: 25355.631 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PRE5, YMR314W, YM9924.06 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40
References: UniProt: P40302, proteasome endopeptidase complex

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Protein/peptide , 3 types, 3 molecules GLs

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


Mass: 27092.719 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PRE10, PRC1, PRS1, YOR362C, O6650 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40
References: UniProt: P21242, proteasome endopeptidase complex
#12: Protein/peptide 26S proteasome subunit RPT4 / 26S protease subunit SUG2 / Proteasomal cap subunit


Mass: 29353.756 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RPT4, CRL13, PCS1, SUG2, YOR259C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P53549
#14: Protein/peptide model substrate polypeptide


Mass: 1572.701 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21

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

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


Mass: 30452.154 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RPT1, CIM5, YTA3, YKL145W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P33299
#9: Protein/peptide 26S proteasome regulatory subunit 4 homolog / Tat-binding homolog 5


Mass: 28889.051 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RPT2, YHS4, YTA5, YDL007W, D2920 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P40327
#10: Protein/peptide 26S proteasome regulatory subunit 8 homolog / Protein CIM3 / Protein SUG1 / Tat-binding protein TBY1


Mass: 29010.736 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RPT6, CIM3, CRL3, SUG1, TBPY, TBY1, YGL048C / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: Q01939
#11: Protein/peptide 26S proteasome regulatory subunit 6B homolog / Protein YNT1 / Tat-binding homolog 2


Mass: 29000.164 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RPT3, YNT1, YTA2, YDR394W, D9509.14 / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P33298
#13: Protein/peptide 26S proteasome regulatory subunit 6A / Tat-binding protein homolog 1 / TBP-1


Mass: 29845.061 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RPT5, YTA1, YOR117W, O3258, YOR3258W / Production host: Saccharomyces cerevisiae S288c (yeast) / Strain (production host): YYS40 / References: UniProt: P33297

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Non-polymers , 2 types, 6 molecules

#15: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (energy-carrying molecule) *YM
#16: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM

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Experimental details

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Experiment

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

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Sample preparation

Component

Type: COMPLEX

IDNameDetailsEntity IDParent IDSource
1Substrate-engaged 26S proteasome in the 5T stateYeast 26S proteasome bound to ubiquitinated substrate in the presence of ATP1,2,3,4,5,6,7,8,9,10,11,12,13,140MULTIPLE SOURCES
2Proteasome1,2,3,4,5,6,7,8,9,10,11,12,131RECOMBINANT
3substrate141RECOMBINANT
Source (natural)
IDEntity assembly IDNcbi tax IDOrganismStrain
12559292Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)ATCC 204508 / S288c
239606Homo sapiens (human)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismStrain
22580240Saccharomyces cerevisiae W303 (yeast)
33511693Escherichia coli BL21 (bacteria)BL21
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer ID
120 mMHEPES1
225 mMNaCl1
325 mMKCl1
410 mMMgCl21
51 mMTCEP1
65 mMATP1
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 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 kelvins
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
MicroscopyMicroscope model: 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 / 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 microns / 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) / Number of grids imaged: 1 / Number of real images: 11656
Image scansSampling size: 5 microns / 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 selectionDetails: Particles were selected using the Relion template-based particle picker
Number of particles selected: 579361
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 47349 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL
Atomic model buildingPDB-ID: 5MPC

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