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- EMDB-5669: Substrate-bound 26S proteasome Rpn11AXA Rpn13-delta mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-5669
TitleSubstrate-bound 26S proteasome Rpn11AXA Rpn13-delta mutant
Map dataReconstruction of substrate-bound Rpn11AXA Rpn13-delta yeast 26S proteasome
Sample
  • Sample: Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome
  • Protein or peptide: 26S proteasomeProteasome
Keywords26S proteasome / 19S regulatory particle / Rpn11 / deubiquitination / AAA+ ATPase / protein translocation / cryoEM / UPS
Function / homologyproteasome regulatory particle / Proteasome, subunit alpha/beta
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsMatyskiela ME / Lander GC / Martin A
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Conformational switching of the 26S proteasome enables substrate degradation.
Authors: Mary E Matyskiela / Gabriel C Lander / Andreas Martin /
Abstract: The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the ...The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains and translocates them into the associated peptidase after unfolding, but detailed mechanisms remain unknown. Here we present the 26S proteasome structure from Saccharomyces cerevisiae during substrate degradation, showing that the regulatory particle switches from a preengaged to a translocation-competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase and a spiral orientation of pore loops that suggests a rapid progression of ATP-hydrolysis events around the ring. Notably, Rpn11 moves from an occluded position to directly above the central pore, thus facilitating substrate deubiquitination concomitant with translocation.
History
DepositionMay 8, 2013-
Header (metadata) releaseMay 22, 2013-
Map releaseJun 19, 2013-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5669.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of substrate-bound Rpn11AXA Rpn13-delta yeast 26S proteasome
Voxel sizeX=Y=Z: 2.17 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-16.684923170000001 - 18.904062270000001
Average (Standard dev.)0.02884262 (±0.63321114)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin404040
Dimensions240240240
Spacing240240240
CellA=B=C: 520.80005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.172.172.17
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z520.800520.800520.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS404040
NC/NR/NS240240240
D min/max/mean-16.68518.9040.029

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Supplemental data

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Sample components

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Entire : Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome

EntireName: Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome
Components
  • Sample: Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome
  • Protein or peptide: 26S proteasomeProteasome

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Supramolecule #1000: Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome

SupramoleculeName: Yeast Rpn11AXA Rpn13-delta mutant 26S proteasome / type: sample / ID: 1000 / Details: mutant proteasome was purified from yeast / Oligomeric state: holoenzyme / Number unique components: 1
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa / Method: Sedimentation

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Macromolecule #1: 26S proteasome

MacromoleculeName: 26S proteasome / type: protein_or_peptide / ID: 1 / Name.synonym: proteasome / Details: Mutant proteasome was purified from yeast / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: yAM11 / synonym: Yeast / Location in cell: cytoplasm
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

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.6
Details: 60mM HEPES, 50mM NaCl, 50mM KCl, 5mM MgCl2, 0.5mM EDTA, 1mM DTT, 2mM ATP, 0.05% NP40
GridDetails: 400-mesh C-flats, 2 um holes with 2 um spacing (Protochips Inc.)
VitrificationCryogen name: ETHANE / Chamber humidity: 50 % / Chamber temperature: 86 K / Instrument: FEI VITROBOT MARK II / Method: Blot 3 seconds with -1 offset

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 100000
Sample stageSpecimen holder: Gatan 626, 70 degree cryoholder / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 78 K / Max: 80 K / Average: 79 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 210,000 times magnification
DetailsData acquired with Leginon
DateSep 10, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 5328 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2/SPARX, FREALIGN
Details: Final map filtered to local resolution using the blocfilt function in Bsoft. Image processing performed in the Appion processing environment.
Number images used: 188400
Details3D reconstruction with EMAN2/SPARX libraries; final alignment of particles with FREALIGN. Sharpened with SPIDER; low-pass filtered to local resolution with BSOFT.

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