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Open data
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Basic information
Entry | Database: PDB / ID: 7thm | |||||||||||||||
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Title | SARS-CoV-2 nsp12/7/8 complex with a native N-terminus nsp9 | |||||||||||||||
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![]() | VIRAL PROTEIN / polymerase / NiRAN / capping / nsp9 | |||||||||||||||
Function / homology | ![]() protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs ...protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / TRAF3-dependent IRF activation pathway / Replication of the SARS-CoV-2 genome / snRNP Assembly / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 5'-3' DNA helicase activity / SARS coronavirus main proteinase / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / host cell endosome / symbiont-mediated suppression of host toll-like receptor signaling pathway / 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 / SARS-CoV-2 modulates host translation machinery / mRNA (guanine-N7)-methyltransferase / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / viral protein processing / lyase activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / RNA helicase / induction by virus of host autophagy / copper ion binding / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / lipid binding / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane Similarity search - Function | |||||||||||||||
Biological species | ![]() ![]() | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å | |||||||||||||||
![]() | Osinski, A. / Tagliabracci, V.S. / Chen, Z. / Li, Y. | |||||||||||||||
Funding support | ![]()
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![]() | ![]() Title: The mechanism of RNA capping by SARS-CoV-2. Authors: Gina J Park / Adam Osinski / Genaro Hernandez / Jennifer L Eitson / Abir Majumdar / Marco Tonelli / Katie Henzler-Wildman / Krzysztof Pawłowski / Zhe Chen / Yang Li / John W Schoggins / ...Authors: Gina J Park / Adam Osinski / Genaro Hernandez / Jennifer L Eitson / Abir Majumdar / Marco Tonelli / Katie Henzler-Wildman / Krzysztof Pawłowski / Zhe Chen / Yang Li / John W Schoggins / Vincent S Tagliabracci / ![]() ![]() ![]() Abstract: The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response. How this cap is made in SARS- ...The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (GpppA) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp14 and nsp16 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19. #1: Journal: Res Sq / Year: 2022 Title: The mechanism of RNA capping by SARS-CoV-2. Authors: Gina J Park / Adam Osinski / Genaro Hernandez / Jennifer L Eitson / Abir Majumdar / Marco Tonelli / Katie Henzler-Wildman / Krzysztof Pawłowski / Zhe Chen / Yang Li / John W Schoggins / ...Authors: Gina J Park / Adam Osinski / Genaro Hernandez / Jennifer L Eitson / Abir Majumdar / Marco Tonelli / Katie Henzler-Wildman / Krzysztof Pawłowski / Zhe Chen / Yang Li / John W Schoggins / Vincent S Tagliabracci / ![]() ![]() Abstract: The SARS-CoV-2 RNA genome contains a 5'-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not ...The SARS-CoV-2 RNA genome contains a 5'-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2'-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19. | |||||||||||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 390.7 KB | Display | ![]() |
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PDB format | ![]() | 317 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 25898MC 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
-Protein , 1 types, 1 molecules A
#1: Protein | Mass: 106780.977 Da / Num. of mol.: 1 / Fragment: UNP residues 4393-5324 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rep, 1a-1b / Production host: ![]() ![]() |
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-Non-structural protein ... , 3 types, 4 molecules BDCG
#2: Protein | Mass: 21903.047 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rep, 1a-1b / Production host: ![]() ![]() #3: Protein | | Mass: 9248.804 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rep, 1a-1b / Production host: ![]() ![]() #4: Protein | | Mass: 12391.171 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rep, 1a-1b / Production host: ![]() ![]() |
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-Non-polymers , 3 types, 4 molecules ![](data/chem/img/ZN.gif)
![](data/chem/img/MN.gif)
![](data/chem/img/POP.gif)
![](data/chem/img/MN.gif)
![](data/chem/img/POP.gif)
#5: Chemical | #6: Chemical | ChemComp-MN / | #7: Chemical | ChemComp-POP / | |
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-Details
Has ligand of interest | Y |
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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: SARS-CoV-2 replication-transcription complex with a native N-terminus nsp9 bound to NiRAN domain of nsp12. Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Value: 0.17203949 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
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: TFS KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39985 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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