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- PDB-7opl: CryoEM structure of DNA Polymerase alpha - primase bound to SARS ... -
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Basic information
Entry | Database: PDB / ID: 7opl | ||||||
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Title | CryoEM structure of DNA Polymerase alpha - primase bound to SARS CoV nsp1 | ||||||
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![]() | DNA BINDING PROTEIN / DNA polymerase / Primase / viral protein | ||||||
Function / homology | ![]() 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 / regulation of type I interferon production / Polymerase switching ...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 / regulation of type I interferon production / Polymerase switching / 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 / primosome complex / 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 / Defective pyroptosis / methyltransferase activity / 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 / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / endonuclease activity / methylation / 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 / virus-mediated perturbation of host defense response / nucleotide binding / 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 | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.12 Å | ||||||
![]() | Kilkenny, M.L. / Pellegrini, L. | ||||||
Funding support | ![]()
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![]() | ![]() 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
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 458.9 KB | Display | ![]() |
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PDB format | ![]() | 359 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 13020MC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA polymerase alpha ... , 2 types, 2 molecules AB
#1: Protein | Mass: 133702.562 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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#2: Protein | Mass: 49855.434 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
-Protein , 3 types, 3 molecules CDE
#3: Protein | Mass: 52590.801 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() References: UniProt: P49642, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases |
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#4: Protein | Mass: 58890.918 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
#5: Protein | Mass: 12955.852 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: 1a / Production host: ![]() ![]() |
-Non-polymers , 2 types, 4 molecules ![](data/chem/img/ZN.gif)
![](data/chem/img/SF4.gif)
![](data/chem/img/SF4.gif)
#6: Chemical | #7: Chemical | ChemComp-SF4 / | |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1 Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT | ||||||||||||||||
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Molecular weight | Value: 0.31 MDa / Experimental value: NO | ||||||||||||||||
Source (natural) | Organism: ![]() | ||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||
Buffer solution | pH: 7.2 | ||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: The nsp1 protein was added in 10-fold stoichiometric excess | ||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: -0.7 nm / Nominal defocus min: -2.5 nm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1.31 sec. / Electron dose: 46.91 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 2919 |
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Processing
Software | Name: PHENIX / Version: 1.19.1_4122: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 709068 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233476 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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