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- EMDB-2594: Deep classification of a large cryo-EM dataset defines the confor... -

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Basic information

Entry
Database: EMDB / ID: EMD-2594
TitleDeep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome
Map dataReconstruction of 26S proteasome in low-energy state (s1)
Sample
  • Sample: 26S Proteasome from Saccharomyces cerevisiaeProteasome
  • Protein or peptide: 26S ProteasomeProteasome
KeywordsProteasome / AAA-ATPase / ATP-analog / classification
Function / homology
Function and homology information


SAGA complex localization to transcription regulatory region / peroxisome fission / proteasome storage granule assembly / transcription export complex 2 / proteasome regulatory particle assembly / protein deneddylation / nonfunctional rRNA decay / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome ...SAGA complex localization to transcription regulatory region / peroxisome fission / proteasome storage granule assembly / transcription export complex 2 / proteasome regulatory particle assembly / protein deneddylation / nonfunctional rRNA decay / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome / proteasome regulatory particle / cytosolic proteasome complex / proteasome regulatory particle, lid subcomplex / proteasome-activating activity / protein-containing complex localization / mitochondrial fission / proteasome regulatory particle, base subcomplex / K48-linked polyubiquitin modification-dependent protein binding / 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 / regulation of protein catabolic process / protein deubiquitination / proteasome storage granule / polyubiquitin modification-dependent protein binding / endopeptidase activator activity / proteasome assembly / positive regulation of RNA polymerase II transcription preinitiation complex assembly / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / enzyme regulator activity / mRNA export from nucleus / : / protein folding chaperone / Neutrophil degranulation / proteasome complex / ubiquitin binding / proteasomal protein catabolic process / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / double-strand break repair via homologous recombination / positive regulation of protein catabolic process / metallopeptidase activity / protein-macromolecule adaptor activity / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / endopeptidase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / molecular adaptor activity / regulation of cell cycle / chromatin remodeling / protein domain specific binding / mRNA binding / ubiquitin protein ligase binding / structural molecule activity / endoplasmic reticulum membrane / endoplasmic reticulum / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / mitochondrion / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Proteasome subunit Rpn10 / Rpn9, C-terminal helix / Rpn9 C-terminal helix / Proteasomal ubiquitin receptor Rpn13/ADRM1 / Proteasomal ubiquitin receptor Rpn13/ADRM1, Pru domain superfamily / Rpn13/ADRM1, Pru domain / Proteasome complex subunit Rpn13, Pru domain / Pru (pleckstrin-like receptor for ubiquitin) domain profile. / 26S proteasome regulatory subunit RPN7/PSMD6 C-terminal helix / 26S proteasome non-ATPase regulatory subunit Rpn12 ...Proteasome subunit Rpn10 / Rpn9, C-terminal helix / Rpn9 C-terminal helix / Proteasomal ubiquitin receptor Rpn13/ADRM1 / Proteasomal ubiquitin receptor Rpn13/ADRM1, Pru domain superfamily / Rpn13/ADRM1, Pru domain / Proteasome complex subunit Rpn13, Pru domain / Pru (pleckstrin-like receptor for ubiquitin) domain profile. / 26S proteasome regulatory subunit RPN7/PSMD6 C-terminal helix / 26S proteasome non-ATPase regulatory subunit Rpn12 / 26S proteasome regulatory subunit, C-terminal / Proteasome regulatory subunit C-terminal / DSS1/SEM1 / 26S proteasome regulatory subunit RPN5, C-terminal domain / : / DSS1/SEM1 family / 26S proteasome regulatory subunit RPN5 C-terminal domain / 26S proteasome subunit RPN2, N-terminal domain / DSS1_SEM1 / 26S proteasome regulatory subunit Rpn6, N-terminal / 6S proteasome subunit Rpn6, C-terminal helix domain / 26S proteasome regulatory subunit RPN6 N-terminal domain / 26S proteasome subunit RPN6 C-terminal helix domain / 26S proteasome regulatory complex, non-ATPase subcomplex, Rpn2/Psmd1 subunit / 26S Proteasome non-ATPase regulatory subunit 13 / 26S proteasome regulatory subunit RPN2, C-terminal / 26S proteasome regulatory subunit RPN2 C-terminal domain / 26S Proteasome non-ATPase regulatory subunit 7/8 / : / 26S proteasome regulatory subunit 7, OB domain / 26S proteasome regulatory complex, non-ATPase subcomplex, Rpn1 subunit / RPN1, N-terminal / 26S proteasome non-ATPase regulatory subunit RPN1, C-terminal / RPN1 N-terminal domain / 26S proteasome non-ATPase regulatory subunit RPN1 C-terminal / 26S proteasome regulatory subunit Rpn7, N-terminal / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome subunit RPN7 / 26S Proteasome non-ATPase regulatory subunit 12/COP9 signalosome complex subunit 4 / Proteasome/cyclosome repeat / Proteasome/cyclosome repeat / PCI/PINT associated module / von Willebrand factor type A domain / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / HEAT repeats / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / PCI domain / Proteasome component (PCI) domain / PCI domain profile. / Ubiquitin interacting motif / Ubiquitin-interacting motif (UIM) domain profile. / 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 / 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. / VWFA domain profile. / Proteasome subunit / Proteasome, subunit alpha/beta / von Willebrand factor, type A / AAA ATPase, AAA+ lid domain / AAA+ lid domain / TPR repeat region circular profile. / ATPase, AAA-type, conserved site / AAA-protein family signature. / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / TPR repeat profile. / MPN domain / MPN domain profile. / Nucleophile aminohydrolases, N-terminal / von Willebrand factor A-like domain superfamily / Tetratricopeptide repeat / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities
Similarity search - Domain/homology
26S proteasome regulatory subunit RPN13 / 26S proteasome complex subunit SEM1 / 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 ...26S proteasome regulatory subunit RPN13 / 26S proteasome complex subunit SEM1 / 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 RPN12 / 26S proteasome regulatory subunit RPN2 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit beta type-1 / 26S proteasome regulatory subunit RPN1 / 26S proteasome regulatory subunit RPN10 / 26S proteasome regulatory subunit RPN3 / 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 / 26S proteasome regulatory subunit RPN9 / 26S proteasome regulatory subunit RPN7 / 26S proteasome regulatory subunit RPN8 / 26S proteasome regulatory subunit RPN5 / 26S proteasome regulatory subunit RPN6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.7 Å
AuthorsUnverdorben P / Beck F / Sledz P / Schweitzer A / Pfeifer G / Plitzko JM / Baumeister W / Foerster F
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome.
Authors: Pia Unverdorben / Florian Beck / Paweł Śledź / Andreas Schweitzer / Günter Pfeifer / Jürgen M Plitzko / Wolfgang Baumeister / Friedrich Förster /
Abstract: The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently ...The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.
History
DepositionFeb 25, 2014-
Header (metadata) releaseMar 26, 2014-
Map releaseApr 2, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.74
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.74
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4cr2
  • Surface level: 0.74
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4cr2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2594.jpg

Downloads & links

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Map

FileDownload / File: emd_2594.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of 26S proteasome in low-energy state (s1)
Voxel sizeX=Y=Z: 1.99 Å
Density
Contour LevelBy AUTHOR: 0.74 / Movie #1: 0.74
Minimum - Maximum-4.11059999 - 6.24226856
Average (Standard dev.)0.00987364 (±0.15704252)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 557.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.991.991.99
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z557.200557.200557.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-4.1116.2420.010

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Supplemental data

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Sample components

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Entire : 26S Proteasome from Saccharomyces cerevisiae

EntireName: 26S Proteasome from Saccharomyces cerevisiaeProteasome
Components
  • Sample: 26S Proteasome from Saccharomyces cerevisiaeProteasome
  • Protein or peptide: 26S ProteasomeProteasome

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Supramolecule #1000: 26S Proteasome from Saccharomyces cerevisiae

SupramoleculeName: 26S Proteasome from Saccharomyces cerevisiae / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.5 MDa

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Macromolecule #1: 26S Proteasome

MacromoleculeName: 26S Proteasome / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Electron beam tilt params: 0
DateDec 24, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 30000 / Average electron dose: 25 e/Å2 / Bits/pixel: 14
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: xmipp / Number images used: 1300000
DetailsThe particles were selected using an automatic selection program. Each physical 26S particles was considered as two particles for processing according to pseudo-C2 symmetry.

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