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- EMDB-7947: Classification of Single Particles from Human Cell Extract Reveal... -

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Entry
Database: EMDB / ID: 7947
TitleClassification of Single Particles from Human Cell Extract Reveals Distinct Structures
Map data3D class of 26S Proteasome
Sample26S Proteasome:
SourceHomo sapiens (human)
Methodsingle particle reconstruction / 31 Å resolution
AuthorsVerbeke EJ / Mallam AL / Drew K / Marcotte EM / Taylor DW
CitationJournal: Cell Rep / Year: 2018
Title: Classification of Single Particles from Human Cell Extract Reveals Distinct Structures.
Authors: Eric J Verbeke / Anna L Mallam / Kevin Drew / Edward M Marcotte / David W Taylor
Abstract: Multi-protein complexes are necessary for nearly all cellular processes, and understanding their structure is required for elucidating their function. Current high-resolution strategies in structural ...Multi-protein complexes are necessary for nearly all cellular processes, and understanding their structure is required for elucidating their function. Current high-resolution strategies in structural biology are effective but lag behind other fields (e.g., genomics and proteomics) due to their reliance on purified samples rather than heterogeneous mixtures. Here, we present a method combining single-particle analysis by electron microscopy with protein identification by mass spectrometry to structurally characterize macromolecular complexes from human cell extract. We identify HSP60 through two-dimensional classification and obtain three-dimensional structures of native proteasomes directly from ab initio classification of a heterogeneous mixture of protein complexes. In addition, we reveal an ∼1-MDa-size structure of unknown composition and reference our proteomics data to suggest possible identities. Our study shows the power of using a shotgun approach to electron microscopy (shotgun EM) when coupled with mass spectrometry as a tool to uncover the structures of macromolecular machines.
DateDeposition: May 29, 2018 / Header (metadata) release: Jun 20, 2018 / Map release: Jul 18, 2018 / Last update: Jul 18, 2018

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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 radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

Fileemd_7947.map.gz (map file in CCP4 format, 11944 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
144 pix
3.6 Å/pix.
= 518.4 Å
144 pix
3.6 Å/pix.
= 518.4 Å
144 pix
3.6 Å/pix.
= 518.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.6 Å
Density
Contour Level:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum-0.031389497 - 0.14382307
Average (Standard dev.)0.0005299318 (0.0066779153)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions144144144
Origin0.0.0.
Limit143.143.143.
Spacing144144144
CellA=B=C: 518.39996 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.63.63.6
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z518.400518.400518.400
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.0310.1440.001

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Supplemental data

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Sample components

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Entire 26S Proteasome

EntireName: 26S Proteasome / Number of components: 1

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Component #1: protein, 26S Proteasome

ProteinName: 26S Proteasome / Recombinant expression: No
MassTheoretical: 2.5 MDa
SourceSpecies: Homo sapiens (human) / Strain: HEK293T

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle
Sample solutionSpecimen conc.: 0.1 mg/ml / pH: 7.4
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2100
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 3 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN MULTISCAN

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Image acquisition

Image acquisitionNumber of digital images: 1500

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 1121
3D reconstructionResolution: 31 Å / Resolution method: FSC 0.143 CUT-OFF

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