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- EMDB-6287: 2.8 Angstrom resolution reconstruction of the T20S proteasome -

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Basic information

Entry
Database: EMDB / ID: EMD-6287
Title2.8 Angstrom resolution reconstruction of the T20S proteasome
Map dataReconstruction of the T20S proteasome at 2.8 Angstrom resolution
Sample
  • Sample: T20S Proteasome
  • Protein or peptide: 20S proteasome
KeywordsDirect detectors / Image processing
Function / homology
Function and homology information


proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / endopeptidase activity / cytoplasm
Similarity search - Function
Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome B-type subunit ...Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal
Similarity search - Domain/homology
Proteasome subunit alpha / Proteasome subunit beta
Similarity search - Component
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsCampbell MG / Veesler D / Cheng A / Potter CS / Carragher B
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
DepositionFeb 22, 2015-
Header (metadata) releaseMar 11, 2015-
Map releaseMar 11, 2015-
UpdateOct 7, 2015-
Current statusOct 7, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bdf
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_6287.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the T20S proteasome at 2.8 Angstrom resolution
Voxel sizeX=Y=Z: 0.982 Å
Density
Contour LevelBy EMDB: 0.025 / Movie #1: 0.03
Minimum - Maximum-0.12976183 - 0.19810915
Average (Standard dev.)0.00023473 (±0.00757627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 294.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9820.9820.982
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z294.600294.600294.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1300.1980.000

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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 proteasome

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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
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.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37313 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 49954

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