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- EMDB-7946: Classification of Single Particles from Human Cell Extract Reveal... -

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Basic information

Entry
Database: EMDB / ID: EMD-7946
TitleClassification of Single Particles from Human Cell Extract Reveals Distinct Structures
Map dataAb initio reconstruction of the proteasome core
Sample
  • Complex: 20 Proteasome Core
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
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.
History
DepositionMay 29, 2018-
Header (metadata) releaseJun 20, 2018-
Map releaseJul 18, 2018-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.21
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.21
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7946.png

Downloads & links

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Map

FileDownload / File: emd_7946.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAb initio reconstruction of the proteasome core
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.21 / Movie #1: 3.21
Minimum - Maximum-0.058355737 - 8.5066805
Average (Standard dev.)0.1261273 (±0.47170013)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 518.39996 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.0588.5070.126

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Supplemental data

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Sample components

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Entire : 20 Proteasome Core

EntireName: 20 Proteasome Core
Components
  • Complex: 20 Proteasome Core

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Supramolecule #1: 20 Proteasome Core

SupramoleculeName: 20 Proteasome Core / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: HEK293T
Molecular weightTheoretical: 750 KDa

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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

Concentration0.1 mg/mL
BufferpH: 7.4
StainingType: NEGATIVE / Material: Uranyl Acetate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 1000.0 nm / Pretreatment - Type: GLOW DISCHARGE
DetailsThe sample was monodisperse.

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Electron microscopy

MicroscopeJEOL 2100
Image recordingFilm or detector model: GATAN MULTISCAN / Number grids imaged: 1 / Number real images: 1500 / Average exposure time: 1.0 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Particle selectionNumber selected: 97000
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 3150
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC

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