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Yorodumi- PDB-7rdy: SARS-CoV-2 replication-transcription complex bound to nsp13 helic... -
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-Basic information
Entry | Database: PDB / ID: 7rdy | ||||||
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Title | SARS-CoV-2 replication-transcription complex bound to nsp13 helicase - nsp13(2)-RTC - engaged class | ||||||
Components |
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Keywords | REPLICATION/TRANSCRIPTION / RNA-dependent RNA polymerase / viral replication-transcription complex / transcription / viral proteins / REPLICATION-TRANSCRIPTION complex | ||||||
Function / homology | Function and homology information protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Lyases; Phosphorus-oxygen lyases / 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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Lyases; Phosphorus-oxygen lyases / 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 / snRNP Assembly / TRAF3-dependent IRF activation pathway / Replication of the SARS-CoV-2 genome / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / : / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / host cell endosome / 3'-5'-RNA exonuclease activity / host cell endoplasmic reticulum-Golgi intermediate compartment / 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 / SARS-CoV-2 modulates host translation machinery / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / DNA helicase / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell endoplasmic reticulum membrane / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / RNA helicase / induction by virus of host autophagy / RNA-directed RNA polymerase / copper ion binding / symbiont-mediated suppression of host gene expression / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / lipid binding / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / 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 | Severe acute respiratory syndrome coronavirus 2 synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Chen, J. / Malone, B. / Campbell, E.A. / Darst, S.A. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Struct Mol Biol / Year: 2022 Title: Ensemble cryo-EM reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex. Authors: James Chen / Qi Wang / Brandon Malone / Eliza Llewellyn / Yakov Pechersky / Kashyap Maruthi / Ed T Eng / Jason K Perry / Elizabeth A Campbell / David E Shaw / Seth A Darst / Abstract: The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA ...The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex or RTC) associated with two copies of nsp13 (nsp13-RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp13-RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp13-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site. #1: Journal: Biorxiv / Year: 2021 Title: Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex Authors: Chen, J. / Wang, Q. / Malone, B. / Llewellyn, E. / Pechersky, Y. / Maruthi, K. / Eng, E.T. / Perry, J.K. / Campbell, E.A. / Shaw, D.E. / Darst, S.A. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7rdy.cif.gz | 485.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7rdy.ent.gz | 400.7 KB | Display | PDB format |
PDBx/mmJSON format | 7rdy.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/rd/7rdy ftp://data.pdbj.org/pub/pdb/validation_reports/rd/7rdy | HTTPS FTP |
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-Related structure data
Related structure data | 24427MC 7rdxC 7rdzC 7re0C 7re1C 7re2C 7re3C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 2 types, 3 molecules AEF
#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.) Severe acute respiratory syndrome coronavirus 2 Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1, RNA-directed RNA polymerase |
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#4: Protein | Mass: 67354.039 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1, DNA helicase, RNA helicase |
-Non-structural protein ... , 2 types, 3 molecules BDC
#2: Protein | Mass: 22034.242 Da / Num. of mol.: 2 / Fragment: UNP residues 3943-4140 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1 #3: Protein | | Mass: 9748.385 Da / Num. of mol.: 1 / Fragment: UNP residues 3860-3942 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTD1 |
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-RNA chain , 2 types, 2 molecules PT
#5: RNA chain | Mass: 11141.644 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#6: RNA chain | Mass: 17573.391 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Non-polymers , 5 types, 19 molecules
#7: Chemical | ChemComp-ZN / #8: Chemical | #9: Chemical | #10: Chemical | #11: Chemical | |
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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 |
-Sample preparation
Component | Name: SARS-CoV-2 replication-transcription complex bound to nsp13 helicase - nsp13(2)-RTC - engaged class Type: COMPLEX / Entity ID: #1-#3, #5-#6 / Source: RECOMBINANT |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Image recording | Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 102615 / Symmetry type: POINT | ||||||||||||||||||||||||
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