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Yorodumi- EMDB-15965: Subtomogram average of SARS-CoV-2 nsp3-4 delate Ubl1-Ubl2 obtaine... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15965 | |||||||||
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Title | Subtomogram average of SARS-CoV-2 nsp3-4 delate Ubl1-Ubl2 obtained from cryo-ET of cryo-FIB milled VeroE6 cells transfected with nsp3-4 delta Ubl1-Ubl2 | |||||||||
Map data | Subtomogram average of SARS-CoV-2 nsp3-4 deltaUbl1-Ubl1 (filtered to 40A) | |||||||||
Sample |
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Keywords | nsp3-4 protein / VIRAL PROTEIN | |||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 30.0 Å | |||||||||
Authors | Chlanda P / Zimmermann L | |||||||||
Funding support | Germany, 1 items
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Citation | Journal: Nat Commun / Year: 2023 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 | emd_15965.map.gz | 19 MB | EMDB map data format | |
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Header (meta data) | emd-15965-v30.xml emd-15965.xml | 13.9 KB 13.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_15965_fsc.xml | 11.9 KB | Display | FSC data file |
Images | emd_15965.png | 21.7 KB | ||
Filedesc metadata | emd-15965.cif.gz | 4.3 KB | ||
Others | emd_15965_half_map_1.map.gz emd_15965_half_map_2.map.gz | 59.3 MB 59.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15965 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15965 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_15965.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Subtomogram average of SARS-CoV-2 nsp3-4 deltaUbl1-Ubl1 (filtered to 40A) | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.156 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_15965_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_15965_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA...
Entire | Name: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA3.1-HA-nsp3-deltaUbl1-Ubl2-nsp4-V5 |
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Components |
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-Supramolecule #1: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA...
Supramolecule | Name: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA3.1-HA-nsp3-deltaUbl1-Ubl2-nsp4-V5 type: cell / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.4 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 42000 |
Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Sample stage | Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |