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- EMDB-1993: Negative stain reconstruction of recombinantly expressed yeast pr... -

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Basic information

Entry
Database: EMDB / ID: EMD-1993
TitleNegative stain reconstruction of recombinantly expressed yeast proteasome lid
Map dataMap of recombinantly expressed Saccharomyces cerevisiae proteasome lid
Sample
  • Sample: recombinantly expressed yeast lid
  • Protein or peptide: lid
Keywords26S / 19S / proteasome / yeast lid / regulatory particle / ubiquitin recognition / deubiquitination / AAA-ATPase
Function / homologyProteasome/cyclosome repeat / proteasome regulatory particle, lid subcomplex
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 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: 4.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

em1993.png

Downloads & links

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Map

FileDownload / File: emd_1993.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of recombinantly expressed Saccharomyces cerevisiae proteasome lid
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.76 Å/pix.
x 128 pix.
= 353.28 Å		2.76 Å/pix.
x 128 pix.
= 353.28 Å		2.76 Å/pix.
x 128 pix.
= 353.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.76 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-4.75049 - 20.128599999999999
Average (Standard dev.)0.00000000735695 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 353.28 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.762.762.76
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z353.280353.280353.280
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-4.75020.1290.000

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Supplemental data

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Sample components

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Entire : recombinantly expressed yeast lid

EntireName: recombinantly expressed yeast lid
Components
  • Sample: recombinantly expressed yeast lid
  • Protein or peptide: lid

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Supramolecule #1000: recombinantly expressed yeast lid

SupramoleculeName: recombinantly expressed yeast lid / type: sample / ID: 1000 / Details: monodisperse / Oligomeric state: 8 subunit complex / Number unique components: 1
Molecular weightExperimental: 361 KDa / Theoretical: 361 KDa / Method: Mass Spectrometry

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Macromolecule #1: lid

MacromoleculeName: lid / type: protein_or_peptide / ID: 1 / Name.synonym: lid / Details: all subunits were recombinantly expressed / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: W303 / synonym: Baker's Yeast
Molecular weightExperimental: 361 KDa / Theoretical: 361 KDa
Recombinant expressionOrganism: Escherichia coli BL21-star (DE3) / Recombinant plasmid: pAM001,pAM002,pAM003
SequenceGO: proteasome regulatory particle, lid subcomplex / InterPro: Proteasome/cyclosome repeat

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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

Concentration0.009 mg/mL
BufferpH: 7.6 / Details: 25mM HEPES, 100mM NaCl, 100mM KCl, 5% glycerol
StainingType: NEGATIVE
Details: Protein adsorbed to grid for 1 minute, then passed over four 50uL drops of 2% w/v uranyl formate, 5 seconds on each drop
GridDetails: 200 mesh Cu grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
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
DateAug 22, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 303 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Room temp single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

DetailsImage processing performed in the Appion processing environment. 3D reconstruction performed using EMAN2 and SPARX libraries
CTF correctionDetails: whole micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 SPARX / Number images used: 29345

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