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Yorodumi- EMDB-13021: CryoEM structure of DNA polymerase alpha - primase bound to SARS ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13021 | |||||||||
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Title | CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein | |||||||||
Map data | 3D refinement map | |||||||||
Sample |
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Function / homology | Function and homology information positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / alpha DNA polymerase:primase complex / Polymerase switching / regulation of type I interferon production ...positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / alpha DNA polymerase:primase complex / Polymerase switching / regulation of type I interferon production / Processive synthesis on the lagging strand / Assembly of the SARS-CoV-1 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Transcription of SARS-CoV-1 sgRNAs / DNA primase activity / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / Translation of Replicase and Assembly of the Replication Transcription Complex / K48-linked deubiquitinase activity / Replication of the SARS-CoV-1 genome / host cell endoplasmic reticulum / K63-linked deubiquitinase activity / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / G1/S-Specific Transcription / leading strand elongation / SARS-CoV-1 modulates host translation machinery / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / viral genome replication / methyltransferase activity / Defective pyroptosis / nuclear matrix / double-strand break repair via nonhomologous end joining / protein import into nucleus / SARS-CoV-1 activates/modulates innate immune responses / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / methylation / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / endonuclease activity / DNA replication / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / DNA-directed DNA polymerase / single-stranded RNA binding / DNA-directed DNA polymerase activity / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA repair / nucleotide binding / virus-mediated perturbation of host defense response / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / proteolysis / DNA binding / zinc ion binding / nucleoplasm / identical protein binding / membrane / nucleus / metal ion binding / cytosol Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.4 Å | |||||||||
Authors | Kilkenny ML / Pellegrini L | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: Protein Sci / Year: 2022 Title: Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α-primase. Authors: Mairi L Kilkenny / Charlotte E Veale / Amir Guppy / Steven W Hardwick / Dimitri Y Chirgadze / Neil J Rzechorzek / Joseph D Maman / Luca Pellegrini / Abstract: The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense ...The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS-CoV-2. The DNA polymerase α (Pol α)-primase complex or primosome-responsible for initiating DNA synthesis during genomic duplication-was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS-CoV-2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo-electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS-CoV-2 interferes with Pol α's putative role in the immune response during the viral infection. | |||||||||
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_13021.map.gz | 11.9 MB | EMDB map data format | |
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Header (meta data) | emd-13021-v30.xml emd-13021.xml | 19.8 KB 19.8 KB | Display Display | EMDB header |
Images | emd_13021.png | 102.7 KB | ||
Others | emd_13021_additional_1.map.gz emd_13021_half_map_1.map.gz emd_13021_half_map_2.map.gz | 14.6 MB 11.9 MB 11.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13021 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13021 | HTTPS FTP |
-Validation report
Summary document | emd_13021_validation.pdf.gz | 456.2 KB | Display | EMDB validaton report |
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Full document | emd_13021_full_validation.pdf.gz | 455.8 KB | Display | |
Data in XML | emd_13021_validation.xml.gz | 9.1 KB | Display | |
Data in CIF | emd_13021_validation.cif.gz | 10.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13021 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13021 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_13021.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D refinement map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.304 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: Sharpened map, calculated from the the two half...
File | emd_13021_additional_1.map | ||||||||||||
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Annotation | Sharpened map, calculated from the the two half maps using local, anisotropic sharpening in Phenix. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map #1
File | emd_13021_half_map_1.map | ||||||||||||
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Annotation | Half map #1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map #2
File | emd_13021_half_map_2.map | ||||||||||||
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Annotation | half map #2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : CryoEM structure of DNA polymerase alpha - primase bound to SARS ...
Entire | Name: CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein |
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Components |
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-Supramolecule #1: CryoEM structure of DNA polymerase alpha - primase bound to SARS ...
Supramolecule | Name: CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Molecular weight | Theoretical: 310 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.3 mg/mL | ||||||||
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Buffer | pH: 7.2 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE | ||||||||
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV | ||||||||
Details | The nsp1 protein was added in 10-fold stoichiometric excess |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 2919 / Average exposure time: 1.31 sec. / Average electron dose: 46.91 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -2.5 µm / Nominal defocus min: -0.7000000000000001 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |