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Entry
Database: EMDB / ID: EMD-2596
TitleDeep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome
Map dataEach physical particles was considered as two particles for processing taking into account the pseudo C2 symmetry
Sample
  • Sample: 26S Proteasome from Saccharomyces cerevisiae
  • Protein or peptide: 26S Proteasome
KeywordsProteasome / AAA-ATPase / ATP-analog / classification
Function / homology
Function and homology information


SAGA complex localization to transcription regulatory region / Metalloprotease DUBs / peroxisome fission / proteasome storage granule assembly / proteasome regulatory particle assembly / transcription export complex 2 / protein deneddylation / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome ...SAGA complex localization to transcription regulatory region / Metalloprotease DUBs / peroxisome fission / proteasome storage granule assembly / proteasome regulatory particle assembly / transcription export complex 2 / protein deneddylation / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome / cytosolic proteasome complex / proteasome regulatory particle / protein-containing complex localization / proteasome regulatory particle, lid subcomplex / proteasome-activating activity / mitochondrial fission / proteasome regulatory particle, base subcomplex / metal-dependent deubiquitinase activity / K48-linked polyubiquitin modification-dependent protein binding / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Proteasome assembly / Cross-presentation of soluble exogenous antigens (endosomes) / TNFR2 non-canonical NF-kB pathway / proteasomal ubiquitin-independent protein catabolic process / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / nonfunctional rRNA decay / Regulation of PTEN stability and activity / CDK-mediated phosphorylation and removal of Cdc6 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / peptide catabolic process / KEAP1-NFE2L2 pathway / Neddylation / proteasome binding / Orc1 removal from chromatin / MAPK6/MAPK4 signaling / regulation of protein catabolic process / proteasome storage granule / Antigen processing: Ubiquitination & Proteasome degradation / polyubiquitin modification-dependent protein binding / protein deubiquitination / positive regulation of RNA polymerase II transcription preinitiation complex assembly / endopeptidase activator activity / proteasome assembly / Ub-specific processing proteases / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / mRNA export from nucleus / enzyme regulator activity / ERAD pathway / Neutrophil degranulation / protein folding chaperone / proteasome complex / ubiquitin binding / positive regulation of transcription elongation by RNA polymerase II / nucleotide-excision repair / double-strand break repair via homologous recombination / positive regulation of protein catabolic process / metallopeptidase activity / peroxisome / ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / molecular adaptor activity / endopeptidase activity / ubiquitinyl hydrolase 1 / proteasome-mediated ubiquitin-dependent protein catabolic process / cysteine-type deubiquitinase activity / regulation of cell cycle / chromatin remodeling / protein domain specific binding / mRNA binding / ubiquitin protein ligase binding / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
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 ...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 / 26S proteasome regulatory subunit RPN5, C-terminal domain / : / 26S proteasome regulatory subunit RPN5 C-terminal domain / PSD13 N-terminal repeats / 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 non-ATPase regulatory subunit 13 / : / 26S proteasome subunit RPN2, N-terminal domain / DSS1/SEM1 / DSS1/SEM1 family / DSS1_SEM1 / 26S proteasome regulatory complex, non-ATPase subcomplex, Rpn2/Psmd1 subunit / 26S proteasome regulatory subunit RPN2, C-terminal / 26S proteasome regulatory subunit RPN2 C-terminal domain / Proteasome subunit Rpn10 / 26S Proteasome non-ATPase regulatory subunit 7/8 / 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 7, OB domain / : / : / : / PSMD12/CSN4, N-terminal / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome regulatory subunit Rpn7, N-terminal / 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 / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / HEAT repeats / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / : / 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 / VWFA domain profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit 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. / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / MPN domain / MPN domain profile. / TPR repeat profile. / Nucleophile aminohydrolases, N-terminal
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: 8.8 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-
UpdateApr 30, 2014-
Current statusApr 30, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.81
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.81
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4cr4
  • Surface level: 0.81
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4cr4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2596.jpg

Downloads & links

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Map

FileDownload / File: emd_2596.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEach physical particles was considered as two particles for processing taking into account the pseudo C2 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.99 Å/pix.
x 280 pix.
= 557.2 Å		1.99 Å/pix.
x 280 pix.
= 557.2 Å		1.99 Å/pix.
x 280 pix.
= 557.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.99 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.81 / Movie #1: 0.81
Minimum - Maximum-3.20770955 - 5.17711926
Average (Standard dev.)0.01165493 (±0.14873062)
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-3.2085.1770.012

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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 cerevisiae
Components
  • Sample: 26S Proteasome from Saccharomyces cerevisiae
  • Protein or peptide: 26S Proteasome

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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
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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.
CTF correctionDetails: micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.8 Å / Resolution method: OTHER / Software - Name: xmipp / Number images used: 560000

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