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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 / 26S Proteasome regulatory subunit 7 / ATPase family associated with various cellular activities (AAA) / 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 4 / Proteasome subunit A N-terminal signature ...AAA+ lid domain / 26S Proteasome regulatory subunit 7 / ATPase family associated with various cellular activities (AAA) / 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 4 / Proteasome subunit A N-terminal signature / Proteasome subunit alpha 1 / Proteasome subunit alpha4 / Proteasome subunit alpha2 / Proteasome subunit alpha5 / Nucleophile aminohydrolases, N-terminal / P-loop containing nucleoside triphosphate hydrolase / Proteasome alpha-type subunit / Proteasome subunit / Proteasome alpha-type subunits signature. / 26S proteasome regulatory subunit P45-like / Ub-specific processing proteases / Antigen processing: Ubiquitination & Proteasome degradation / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / rt:r-sce-69229: / CDK-mediated phosphorylation and removal of Cdc6 / Orc1 removal from chromatin / Neutrophil degranulation / MAPK6/MAPK4 signaling / AAA-protein family signature. / TNFR2 non-canonical NF-kB pathway / 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. / Proteasome beta-type subunit, conserved site / Proteasome subunit alpha6 / ATPase, AAA-type, conserved site / AAA+ ATPase domain / Proteasome, subunit alpha/beta / Proteasome alpha-subunit, N-terminal domain / ATPase, AAA-type, core / protein-containing complex localization / proteasome regulatory particle assembly / nonfunctional rRNA decay / proteasome-activating ATPase activity / nuclear proteasome complex / proteasome regulatory particle, base subcomplex / cytosolic proteasome complex / peptide catabolic process / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / proteasomal ubiquitin-independent protein catabolic process / proteasome storage granule / proteasome endopeptidase complex / proteasome core complex, alpha-subunit complex / ubiquitin-dependent ERAD pathway / threonine-type endopeptidase activity / TBP-class protein binding / positive regulation of transcription elongation from RNA polymerase II promoter / 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 / ATPase activity / mRNA binding / protein domain specific 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 / A Martin
Abstract: The 26 proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric ATPase motor of the proteasome unfolds and ...The 26 proteasome is the primary eukaryotic degradation machine and thus critically involved in numerous cellular processes. The hetero-hexameric 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-EM 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+ 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

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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 mm / 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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