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- EMDB-6464: Reconstruction of the T20S proteasome at 2.8 Angstrom resolution ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6464
TitleReconstruction of the T20S proteasome at 2.8 Angstrom resolution using optimal exposure filtering
Map dataUnsharpened map of the T20S Proteasome obtained with exposure filtered particles and Frealign
Sample
  • Sample: T20S Proteasome
  • Protein or peptide: 20S proteasomeProteasome
Keywordsexposure filter / dose / image processing
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsGrant T / Grigorieff N
CitationJournal: Elife / Year: 2015
Title: 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy.
Authors: Melody G Campbell / David Veesler / Anchi Cheng / Clinton S Potter / Bridget Carragher /
Abstract: Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ...Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ∼3.3 Å) reconstructions. Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 Å resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design.
History
DepositionSep 10, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseSep 23, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00965
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.00965
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.00965
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6464.gif

Downloads & links

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Map

FileDownload / File: emd_6464.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map of the T20S Proteasome obtained with exposure filtered particles and Frealign
Voxel sizeX=Y=Z: 0.982 Å
Density
Contour LevelBy AUTHOR: 0.00965 / Movie #1: 0.00965
Minimum - Maximum-0.00940228 - 0.02921085
Average (Standard dev.)-0.00052495 (±0.00164555)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 314.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9820.9820.982
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z314.240314.240314.240
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-180
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0090.029-0.001

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Supplemental data

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Sample components

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Entire : T20S Proteasome

EntireName: T20S Proteasome
Components
  • Sample: T20S Proteasome
  • Protein or peptide: 20S proteasomeProteasome

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Supramolecule #1000: T20S Proteasome

SupramoleculeName: T20S Proteasome / type: sample / ID: 1000 / Oligomeric state: D7 / Number unique components: 1
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: 20S proteasome

MacromoleculeName: 20S proteasome / type: protein_or_peptide / ID: 1 / Number of copies: 28 / Oligomeric state: 28-mer / Recombinant expression: Yes
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pREAR-A

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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

Concentration0.21 mg/mL
BufferpH: 7.8 / Details: 20 mM Tris, 150 mM NaCl
GridDetails: 1.2/1.3 C-Flat grid, plasma-cleaned
VitrificationCryogen name: ETHANE / Chamber temperature: 95 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Vitrification carried out at room temperature. / Method: Blot for 2.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37313 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 22,500 times magnification.
DateSep 5, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 192 / Average electron dose: 53 e/Å2
Details: Each movie was acquired over 7.6 seconds and comprises 38 frames.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF parameters provided by Campbell et al.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: OTHER / Software - Name: Tigris, Unblur, Frealign
Details: Map was created from exposure-filtered images. No information from resolutions greater than 5A was used in the refinement.
Number images used: 49954
DetailsParticles were exposure-filtered then refined and reconstructed using Frealign.

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