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- EMDB-2656: The 15S precursor complex of the yeast 20S proteasome -

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Basic information

Entry
Database: EMDB / ID: EMD-2656
TitleThe 15S precursor complex of the yeast 20S proteasome
Map data3D reconstruction of the 15S proteasomal precursor complex from S. cerevisiae
Sample
  • Sample: 15S proteasomal precursor complex from S. cerevisiae containing the dedicated assembly chaperones Pba1, Pba2 and Ump1
  • Protein or peptide: 15S proteasomal precursor complex
Keywords15S precursor / Pba1 / Pba2 / proteasome / yeast
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 19.3 Å
AuthorsKock M / Nunes MM / Hemann M / Kube S / Dohmen JR / Herzog F / Ramos PC / Wendler P
CitationJournal: Nat Commun / Year: 2015
Title: Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone.
Authors: Malte Kock / Maria M Nunes / Matthias Hemann / Sebastian Kube / R Jürgen Dohmen / Franz Herzog / Paula C Ramos / Petra Wendler /
Abstract: The chaperones Ump1 and Pba1-Pba2 promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta7. Here we ...The chaperones Ump1 and Pba1-Pba2 promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta7. Here we elucidate the structural role of these chaperones in late steps of core particle biogenesis using biochemical, electron microscopy, cross-linking and mass spectrometry analyses. In 15S precursor complexes, Ump1 is largely unstructured, lining the inner cavity of the complex along the interface between alpha and beta subunits. The alpha and beta subunits form loosely packed rings with a wider alpha ring opening than in the 20S core particle, allowing for the Pba1-Pba2 heterodimer to be partially embedded in the central alpha ring cavity. During biogenesis, the heterodimer is expelled from the alpha ring by a restructuring event that organizes the beta ring and leads to tightening of the alpha ring opening. In this way, the Pba1-Pba2 chaperone is recycled for a new round of proteasome assembly.
History
DepositionMay 20, 2014-
Header (metadata) releaseJun 18, 2014-
Map releaseMar 11, 2015-
UpdateMar 11, 2015-
Current statusMar 11, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2656.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of the 15S proteasomal precursor complex from S. cerevisiae
Voxel sizeX=Y=Z: 2.9 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-2.20301461 - 9.96145725
Average (Standard dev.)0.02990587 (±0.3000105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 290.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.92.92.9
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z290.000290.000290.000
α/β/γ90.00090.00090.000
start NX/NY/NZ0-51-100
NX/NY/NZ82103201
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-2.2039.9610.030

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Supplemental data

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Sample components

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Entire : 15S proteasomal precursor complex from S. cerevisiae containing t...

EntireName: 15S proteasomal precursor complex from S. cerevisiae containing the dedicated assembly chaperones Pba1, Pba2 and Ump1
Components
  • Sample: 15S proteasomal precursor complex from S. cerevisiae containing the dedicated assembly chaperones Pba1, Pba2 and Ump1
  • Protein or peptide: 15S proteasomal precursor complex

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Supramolecule #1000: 15S proteasomal precursor complex from S. cerevisiae containing t...

SupramoleculeName: 15S proteasomal precursor complex from S. cerevisiae containing the dedicated assembly chaperones Pba1, Pba2 and Ump1
type: sample / ID: 1000 / Oligomeric state: Monomer / Number unique components: 1
Molecular weightExperimental: 440 KDa / Theoretical: 440 KDa / Method: Sedimentation

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Macromolecule #1: 15S proteasomal precursor complex

MacromoleculeName: 15S proteasomal precursor complex / type: protein_or_peptide / ID: 1 / Name.synonym: 15S / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: MO27 / synonym: Bakers' Yeast
Molecular weightExperimental: 440 KDa / Theoretical: 440 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 7.5
Details: 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM ATP, 150 mM NaCl, 15 % (v/v) glycerol, 10 mM imidazole
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 4 drops of 2 % (w/v) uranyl acetate for 10 s each
GridDetails: 400 mesh copper grid with carbon support, glow discharged in ambient atmosphere
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 103448
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 96000 times magnification
DateApr 30, 2012
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (2k x 2k) / Number real images: 218 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Micrograph-based phase flipping
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.3 Å / Resolution method: OTHER / Software - Name: IMAGIC, Spider / Number images used: 10156
DetailsParticles were selected manually

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