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- EMDB-15926: Cryo-electron tomogram of VeroE6 cells transfected with HA-nsp3-d... -
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Open data
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Basic information
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Title | Cryo-electron tomogram of VeroE6 cells transfected with HA-nsp3-deltaUbl1-Ubl2-nsp4-V5, plunge frozen at 16 hpt | |||||||||
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![]() | complex / VIRAL PROTEIN | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | electron tomography / cryo EM | |||||||||
![]() | Zimmermann L / Chlanda P | |||||||||
Funding support | ![]()
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![]() | ![]() Title: SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle. Authors: Liv Zimmermann / Xiaohan Zhao / Jana Makroczyova / Moritz Wachsmuth-Melm / Vibhu Prasad / Zach Hensel / Ralf Bartenschlager / Petr Chlanda / ![]() ![]() Abstract: Coronavirus replication is associated with the remodeling of cellular membranes, resulting in the formation of double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal ...Coronavirus replication is associated with the remodeling of cellular membranes, resulting in the formation of double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal for viral RNA. However, the exact components and the structure of the SARS-CoV-2 DMV pore remain to be determined. Here, we investigate the structure of the DMV pore by in situ cryo-electron tomography combined with subtomogram averaging. We identify non-structural protein (nsp) 3 and 4 as minimal components required for the formation of a DMV-spanning pore, which is dependent on nsp3-4 proteolytic cleavage. In addition, we show that Mac2-Mac3-DPUP-Ubl2 domains are critical for nsp3 oligomerization and crown integrity which influences membrane curvature required for biogenesis of DMVs. Altogether, SARS-CoV-2 nsp3-4 have a dual role by driving the biogenesis of replication organelles and assembly of DMV-spanning pores which we propose here to term replicopores. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 2.2 GB | ![]() | |
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Header (meta data) | ![]() ![]() | 8.4 KB 8.4 KB | Display Display | ![]() |
Images | ![]() | 159.4 KB | ||
Filedesc metadata | ![]() | 3.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 417.9 KB | Display | ![]() |
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Full document | ![]() | 417.5 KB | Display | |
Data in XML | ![]() | 4.6 KB | Display | |
Data in CIF | ![]() | 5.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 6.468 Å | ||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : Severe acute respiratory syndrome coronavirus 2
Entire | Name: ![]() ![]() |
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Components |
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-Supramolecule #1: Severe acute respiratory syndrome coronavirus 2
Supramolecule | Name: Severe acute respiratory syndrome coronavirus 2 / type: virus / ID: 1 / Parent: 0 / Details: nsp3-4 gene / NCBI-ID: 2697049 Sci species name: Severe acute respiratory syndrome coronavirus 2 Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: Yes |
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-Experimental details
-Structure determination
Method | cryo EM |
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![]() | electron tomography |
Aggregation state | cell |
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Sample preparation
Buffer | pH: 7.4 |
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Vitrification | Cryogen name: ETHANE |
Cryo protectant | NO |
Sectioning | Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 200 / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 200 Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is Aquilos. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Number images used: 41 |
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