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- EMDB-1992: The Saccharomyces cerevisiae 26S proteasome at subnanometer resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-1992
TitleThe Saccharomyces cerevisiae 26S proteasome at subnanometer resolution
Map data26 proteasome
Sample
  • Sample: 26S ProteasomeProteasome
  • Protein or peptide: 26S ProteasomeProteasome
Keywords26S / 19S / proteasome / regulatory particle / ubiquitin recognition / deubiquitination / AAA-ATPase
Function / homologyproteasome regulatory particle / Proteasome, subunit alpha/beta
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.0 Å
AuthorsLander GC / Estrin E / Matyskiela M / Bashore C / Nogales E / Martin A
CitationJournal: Nature / Year: 2012
Title: Complete subunit architecture of the proteasome regulatory particle.
Authors: Gabriel C Lander / Eric Estrin / Mary E Matyskiela / Charlene Bashore / Eva Nogales / Andreas Martin /
Abstract: The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, ...The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate's path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.
History
DepositionNov 18, 2011-
Header (metadata) releaseJan 6, 2012-
Map releaseJan 6, 2012-
UpdateFeb 3, 2012-
Current statusFeb 3, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_1992.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation26 proteasome
Voxel sizeX=Y=Z: 2.17 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-21.591999999999999 - 33.732799999999997
Average (Standard dev.)0.0686551 (±1.12869)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin161616
Dimensions256256256
Spacing256256256
CellA=B=C: 555.52 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.172.172.17
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z555.520555.520555.520
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS161616
NC/NR/NS256256256
D min/max/mean-21.59233.7330.069

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Supplemental data

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Sample components

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Entire : 26S Proteasome

EntireName: 26S ProteasomeProteasome
Components
  • Sample: 26S ProteasomeProteasome
  • Protein or peptide: 26S ProteasomeProteasome

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Supramolecule #1000: 26S Proteasome

SupramoleculeName: 26S Proteasome / type: sample / ID: 1000 / Details: monodisperse / Oligomeric state: holoenzyme / Number unique components: 1
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa / Method: Mass Spectrometry

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Macromolecule #1: 26S Proteasome

MacromoleculeName: 26S Proteasome / type: protein_or_peptide / ID: 1 / Name.synonym: 26S Proteasome / Details: Endogenous proteasome was purified from yeast / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YYS40 / synonym: Yeast
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa
SequenceGO: proteasome regulatory particle / InterPro: Proteasome, subunit alpha/beta

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.7 mg/mL
BufferpH: 7.6
Details: 20mM HEPES, 50mM NaCl, 50mM KCl, 1mM ATP, 1mM DTT, 0.05% NP40
StainingType: NEGATIVE
Details: C-flat grids made hydrophilic with Solarus Plasma cleaner, 4 uL of sample applied, blotted in Vitrobot for 3 seconds with offset -1, plunged into liquid ethane
GridDetails: 400-mesh C-flats, 2um holes with 2um spacing (Protochips Inc.)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 86 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot 3 seconds with -2 offset

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 100000
Sample stageSpecimen holder: Side-entry cryostage / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 78 K / Max: 78 K / Average: 78 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 210,000 times magnification
Legacy - Electron beam tilt params: 0
DetailsData acquired using Leginon
DateSep 11, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 9153 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 SPARX
Details: Final map filtered to local resolution using the blocfilt function in Bsoft
Number images used: 93679
DetailsImage processing performed in the Appion processing environment. 3D reconstruction performed using EMAN2 and SPARX libraries

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