|Entry||Database: EMDB / ID: 7947|
|Title||Classification of Single Particles from Human Cell Extract Reveals Distinct Structures|
|Map data||3D class of 26S Proteasome|
|Source||Homo sapiens (human)|
|Method||single particle reconstruction / 31 Å resolution|
|Authors||Verbeke EJ / Mallam AL / Drew K / Marcotte EM / Taylor DW|
|Citation||Journal: 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.
|Date||Deposition: May 29, 2018 / Header (metadata) release: Jun 20, 2018 / Map release: Jul 18, 2018 / Last update: Jul 18, 2018|
|Structure viewer||EM map: |
Downloads & links
|File||emd_7947.map.gz (map file in CCP4 format, 11944 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 3.6 Å|
CCP4 map header:
-Entire 26S Proteasome
|Entire||Name: 26S Proteasome / Number of components: 1|
-Component #1: protein, 26S Proteasome
|Protein||Name: 26S Proteasome / Recombinant expression: No|
|Mass||Theoretical: 2.5 MDa|
|Source||Species: Homo sapiens (human) / Strain: HEK293T|
|Specimen||Specimen state: particle|
|Sample solution||Specimen conc.: 0.1 mg/ml / pH: 7.4|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
|Imaging||Microscope: JEOL 2100|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 3 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN MULTISCAN|
|Image acquisition||Number of digital images: 1500|
|Processing||Method: single particle reconstruction / Number of projections: 1121|
|3D reconstruction||Resolution: 31 Å / Resolution method: FSC 0.143 CUT-OFF|
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