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Yorodumi- EMDB-7947: Classification of Single Particles from Human Cell Extract Reveal... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-7947 | |||||||||
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Title | Classification of Single Particles from Human Cell Extract Reveals Distinct Structures | |||||||||
Map data | 3D class of 26S Proteasome | |||||||||
Sample |
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Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 31.0 Å | |||||||||
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. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_7947.map.gz | 3.5 MB | EMDB map data format | |
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Header (meta data) | emd-7947-v30.xml emd-7947.xml | 9 KB 9 KB | Display Display | EMDB header |
Images | emd_7947.png | 40.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-7947 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-7947 | HTTPS FTP |
-Validation report
Summary document | emd_7947_validation.pdf.gz | 77.9 KB | Display | EMDB validaton report |
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Full document | emd_7947_full_validation.pdf.gz | 77 KB | Display | |
Data in XML | emd_7947_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-7947 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-7947 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_7947.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D class of 26S Proteasome | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.6 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : 26S Proteasome
Entire | Name: 26S Proteasome |
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Components |
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-Supramolecule #1: 26S Proteasome
Supramolecule | Name: 26S Proteasome / type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Homo sapiens (human) / Strain: HEK293T |
Molecular weight | Theoretical: 2.5 MDa |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 7.4 |
Staining | Type: NEGATIVE / Material: Uranyl Acetate |
Grid | Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 1000.0 nm |
Details | The sample was monodisperse. |
-Electron microscopy
Microscope | JEOL 2100 |
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Image recording | Film 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 beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |