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- PDB-7re3: SARS-CoV-2 replication-transcription complex bound to nsp13 helic... -

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Basic information

Entry
Database: PDB / ID: 7re3
TitleSARS-CoV-2 replication-transcription complex bound to nsp13 helicase - nsp13(2)-RTC dimer
Components
  • (Non-structural protein ...Viral nonstructural protein) x 2
  • Helicase
  • Product RNA
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
  • Template RNA
KeywordsREPLICATION/TRANSCRIPTION / RNA-dependent RNA polymerase / viral replication-transcription complex / transcription / viral proteins / REPLICATION-TRANSCRIPTION complex
Function / homology
Function and homology information


Maturation of replicase proteins / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / exoribonuclease complex / viral RNA-directed RNA polymerase complex / endoribonuclease complex / Transcription of SARS-CoV-2 sgRNAs / endopeptidase complex / 5'-3' RNA helicase activity / mRNA cap methyltransferase complex ...Maturation of replicase proteins / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / exoribonuclease complex / viral RNA-directed RNA polymerase complex / endoribonuclease complex / Transcription of SARS-CoV-2 sgRNAs / endopeptidase complex / 5'-3' RNA helicase activity / mRNA cap methyltransferase complex / RNA phosphodiester bond hydrolysis, exonucleolytic / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-2 genome / positive regulation of RNA biosynthetic process / Lyases; Phosphorus-oxygen lyases / modulation by virus of host autophagy / mRNA methylation / double membrane vesicle viral factory outer membrane / suppression by virus of host translation / ISG15-specific peptidase activity / SARS coronavirus main proteinase / suppression by virus of host type I interferon production / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 3'-5'-exoribonuclease activity / host cell endosome / host cell endoplasmic reticulum-Golgi intermediate compartment / induction by virus of catabolism of host mRNA / cytoplasmic viral factory / protein K48-linked deubiquitination / G-quadruplex RNA binding / suppression by virus of host ISG15-protein conjugation / 7-methylguanosine mRNA capping / transcription, RNA-templated / viral transcription / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / host cell Golgi apparatus / suppression by virus of host toll-like receptor signaling pathway / host cell endoplasmic reticulum / suppression by virus of host NF-kappaB cascade / modulation by virus of host protein ubiquitination / protein K63-linked deubiquitination / methyltransferase cap1 / positive regulation of viral genome replication / mRNA (nucleoside-2'-O-)-methyltransferase activity / positive stranded viral RNA replication / suppression by virus of host TRAF activity / protein autoprocessing / cysteine-type peptidase activity / helicase activity / ubiquitinyl hydrolase 1 / DNA helicase / DNA helicase activity / cysteine-type deubiquitinase activity / single-stranded RNA binding / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / protein processing / host cell perinuclear region of cytoplasm / viral protein processing / RNA helicase / lyase activity / induction by virus of host autophagy / RNA-directed RNA polymerase / suppression by virus of host gene expression / endonuclease activity / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / transcription, DNA-templated / suppression by virus of host type I interferon-mediated signaling pathway / host cell cytoplasm / protein dimerization activity / host cell nucleus / ATP hydrolysis activity / protein homodimerization activity / zinc ion binding / integral component of membrane / ATP binding / identical protein binding / cytosol
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nonstructural protein 14, betacoronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nonstructural protein 14, betacoronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Viral (Superfamily 1) RNA helicase / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Non-structural protein 14, coronavirus / Non-structural protein NSP16, coronavirus-like / RNA polymerase, N-terminal, coronavirus / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Coronavirus RNA-dependent RNA polymerase, N-terminal / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerisation / Nonstructural protein 15, middle domain, coronavirus / Coronavirus replicase NSP15, N-terminal oligomerisation / Coronavirus replicase NSP15, middle domain / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / Endoribonuclease EndoU-like / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Betacoronavirus replicase NSP3, N-terminal / Non-structural protein 2, SARS-CoV-like / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP1 superfamily, betacoronavirus / Non-structural protein NSP3, N-terminal, betacoronavirus / Betacoronavirus SUD-C domain / (+)RNA virus helicase core domain profile. / (+) RNA virus helicase core domain / Carbamoyl-phosphate synthase subdomain signature 2. / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Lipocalin signature. / Coronavirus (CoV) Nsp1 globular domain profile. / Betacoronavirus Nsp3c-M domain profile. / Betacoronavirus single-stranded poly(A) binding domain / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Betacoronavirus replicase NSP1 / Non-structural protein NSP1, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Non-structural protein 6, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / Coronavirus replicase NSP2, N-terminal / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus Nsp3d Ubl domain profile. / Coronavirus Nsp3a Ubl domain profile. / Coronavirus replicase NSP2, C-terminal / Non-structural protein 2, C-terminal domain, coronavirus / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Papain-like protease, thumb domain superfamily, coronavirus / Non-structural protein NSP7, coronavirus / Peptidase family C16 domain profile. / Coronavirus replicase NSP7 / Coronavirus papain-like peptidase / Coronavirus endopeptidase C30 / Coronavirus replicase NSP8 / Coronavirus RNA synthesis protein NSP10 / Coronavirus replicase NSP4, C-terminal / Coronavirus replicase NSP3, C-terminal / Coronavirus replicase NSP6 / Coronavirus replicase NSP4, N-terminal / Coronavirus replicase NSP3, C-terminal / Coronavirus main protease (M-pro) domain profile. / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP4, N-terminal, coronavirus / Non-structural protein 6, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP8 superfamily, coronavirus / Non-structural protein NSP7 superfamily, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus / RNA synthesis protein NSP10, coronavirus
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / RNA (> 10) / RNA / ALUMINUM FLUORIDE / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsChen, J. / Malone, B. / Campbell, E.A. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
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
DepositionJul 12, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 24, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author / Item: _citation.title
Revision 1.2Mar 23, 2022Group: Database references / Category: citation / citation_author
Revision 1.3Mar 30, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: RNA-directed RNA polymerase
B: Non-structural protein 8
C: Non-structural protein 7
D: Non-structural protein 8
E: Helicase
F: Helicase
H: Non-structural protein 8
P: Product RNA
T: Template RNA
G: RNA-directed RNA polymerase
I: Non-structural protein 7
J: Non-structural protein 8
K: Helicase
L: Helicase
Q: Product RNA
U: Template RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)653,39750
Polymers648,04216
Non-polymers5,35534
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 6 molecules AGEFKL

#1: Protein RNA-directed RNA polymerase / RNA-dependent RNA polymerase / Pol / RdRp / Non-structural protein 12 / nsp12


Mass: 106780.977 Da / Num. of mol.: 2 / 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
#4: Protein
Helicase / / Hel / Non-structural protein 13 / nsp13


Mass: 67354.039 Da / Num. of mol.: 4 / Fragment: UNP residues 5325-5925
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

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Non-structural protein ... , 2 types, 6 molecules BDHJCI

#2: Protein
Non-structural protein 8 / nsp8


Mass: 22034.242 Da / Num. of mol.: 4 / 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 Non-structural protein 7 / nsp7


Mass: 9748.385 Da / Num. of mol.: 2 / 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, 4 molecules PQTU

#5: RNA chain Product RNA


Mass: 11141.644 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: RNA chain Template RNA


Mass: 17573.391 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 5 types, 34 molecules

#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Zn
#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#9: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#10: Chemical
ChemComp-AF3 / ALUMINUM FLUORIDE / Aluminium fluoride


Mass: 83.977 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: AlF3
#11: Chemical ChemComp-1N7 / CHAPSO / 2-hydroxy-N,N-dimethyl-3-sulfo-N-(3-{[(3beta,5beta,7beta,12beta)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}propyl)propan-1-aminium / CHAPS detergent


Mass: 631.884 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H59N2O8S / Comment: detergent*YM

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - nsp13(2)-RTC dimer
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35392 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00444358
ELECTRON MICROSCOPYf_angle_d0.99760983
ELECTRON MICROSCOPYf_dihedral_angle_d9.2947349
ELECTRON MICROSCOPYf_chiral_restr0.0497092
ELECTRON MICROSCOPYf_plane_restr0.0087223

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