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- PDB-7krn: Structure of SARS-CoV-2 backtracked complex bound to nsp13 helica... -

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Basic information

Entry
Database: PDB / ID: 7krn
TitleStructure of SARS-CoV-2 backtracked complex bound to nsp13 helicase - nsp13(1)-BTC
Components
  • (Non-structural protein ...Viral nonstructural protein) x 2
  • Helicase
  • RNA (37-MER)
  • RNA (43-MER)
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsTRANSFERASE/HYDROLASE/RNA / RNA-dependent RNA polymerase / viral replication-transcription complex / transcription / viral proteins / TRANSFERASE-HYDROLASE-RNA complex
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / viral RNA-directed RNA polymerase complex / exoribonuclease complex / viral replication complex formation and maintenance / 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 / endopeptidase complex / endoribonuclease complex / Maturation of replicase proteins ...protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / viral RNA-directed RNA polymerase complex / exoribonuclease complex / viral replication complex formation and maintenance / 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 / endopeptidase complex / endoribonuclease complex / Maturation of replicase proteins / mRNA cap methyltransferase complex / : / 5'-3' RNA helicase activity / mRNA cap binding complex / positive regulation of RNA biosynthetic process / Lyases; Phosphorus-oxygen lyases / Translation of Replicase and Assembly of the Replication Transcription Complex / mRNA methylation / modulation by virus of host autophagy / Replication of the SARS-CoV-2 genome / Transcription of SARS-CoV-2 sgRNAs / double membrane vesicle viral factory outer membrane / suppression by virus of host translation / ISG15-specific peptidase activity / snRNP Assembly / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / SARS coronavirus main proteinase / TRAF3-dependent IRF activation pathway / suppression by virus of host type I interferon production / host cell endosome / mRNA guanylyltransferase / cytoplasmic viral factory / induction by virus of catabolism of host mRNA / protein K48-linked deubiquitination / 3'-5'-RNA exonuclease activity / SARS-CoV-2 modulates host translation machinery / G-quadruplex RNA binding / suppression by virus of host ISG15-protein conjugation / host cell endoplasmic reticulum-Golgi intermediate compartment / omega peptidase activity / RNA-templated transcription / 7-methylguanosine mRNA capping / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / viral transcription / suppression by virus of host NF-kappaB cascade / protein K63-linked deubiquitination / suppression by virus of host toll-like receptor signaling pathway / mRNA (guanine-N7)-methyltransferase / modulation by symbiont of host protein ubiquitination / host cell endoplasmic reticulum / methyltransferase cap1 / host cell Golgi apparatus / disruption by virus of host TRAF-mediated signal transduction / positive regulation of viral genome replication / positive stranded viral RNA replication / protein autoprocessing / mRNA (nucleoside-2'-O-)-methyltransferase activity / cysteine-type peptidase activity / mRNA (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / DNA helicase / cysteine-type deubiquitinase activity / helicase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / protein processing / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / RNA helicase / induction by virus of host autophagy / RNA-directed RNA polymerase / suppression by virus of host gene expression / copper ion binding / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-dependent RNA polymerase activity / host cell cytoplasm / protein dimerization activity / DNA-templated transcription / lipid binding / suppression by virus of host type I interferon-mediated signaling pathway / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / protein homodimerization activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane / identical protein binding
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Coronavirus Nsp12 Interface domain profile. / Nonstructural protein 14, betacoronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP15, NendoU domain, coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / NSP12 RNA-dependent RNA polymerase, coronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Coronavirus Nsp12 Interface domain profile. / Nonstructural protein 14, betacoronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP15, NendoU domain, coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / NSP12 RNA-dependent RNA polymerase, coronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 2-O-methyltransferase / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Nonstructural protein 13, 1B domain, coronavirus / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerization / Nonstructural protein 15, middle domain, coronavirus / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Non-structural protein NSP16, coronavirus-like / Coronavirus 2'-O-methyltransferase / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Viral (Superfamily 1) RNA helicase / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / NSP1 globular domain superfamily, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / Lipocalin signature. / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / Carbamoyl-phosphate synthase subdomain signature 2. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 middle domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Coronavirus Nsp3 Y3 domain profile. / Betacoronavirus Nsp3c-M domain profile. / NSP1, C-terminal domain, betacoronavirus / NSP1, globular domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Papain-like protease, N-terminal domain superfamily, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, N-terminal / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Papain-like protease, thumb domain superfamily, coronavirus / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Non-structural protein NSP7, coronavirus / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP8, coronavirus / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ALUMINUM FLUORIDE / RNA / RNA (> 10) / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2 (unknown)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsChen, J. / Malone, B. / Campbell, E.A. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex.
Authors: Brandon Malone / James Chen / Qi Wang / Eliza Llewellyn / Young Joo Choi / Paul Dominic B Olinares / Xinyun Cao / Carolina Hernandez / Edward T Eng / Brian T Chait / David E Shaw / Robert ...Authors: Brandon Malone / James Chen / Qi Wang / Eliza Llewellyn / Young Joo Choi / Paul Dominic B Olinares / Xinyun Cao / Carolina Hernandez / Edward T Eng / Brian T Chait / David E Shaw / Robert Landick / Seth A Darst / Elizabeth A Campbell /
Abstract: Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not ...Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.
History
DepositionNov 20, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 21, 2021Provider: repository / Type: Initial release
Revision 1.1May 5, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_DOI ..._citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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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
P: RNA (37-MER)
T: RNA (43-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,40320
Polymers258,1937
Non-polymers3,21013
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AE

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


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 (unknown)
Gene: rep, 1a-1b / Production host: Escherichia coli K-12 (unknown) / References: UniProt: P0DTD1, RNA-directed RNA polymerase
#4: Protein Helicase / / Hel / Non-structural protein 13 / nsp13


Mass: 67354.039 Da / Num. of mol.: 1 / Fragment: UNP residues 5325-5925
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 (unknown)
Gene: rep, 1a-1b / Production host: Escherichia coli K-12 (unknown) / References: UniProt: P0DTD1, DNA helicase, RNA helicase

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Non-structural protein ... , 2 types, 3 molecules BDC

#2: Protein Non-structural protein 8 / nsp8


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 (unknown)
Gene: rep, 1a-1b / Production host: Escherichia coli K-12 (unknown) / References: UniProt: P0DTD1
#3: Protein Non-structural protein 7 / nsp7


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 (unknown)
Gene: rep, 1a-1b / Production host: Escherichia coli K-12 (unknown) / References: UniProt: P0DTD1

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RNA chain , 2 types, 2 molecules PT

#5: RNA chain RNA (37-MER)


Mass: 12667.554 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2 (unknown)
#6: RNA chain RNA (43-MER)


Mass: 17573.391 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2 (unknown)

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Non-polymers , 5 types, 13 molecules

#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#10: 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.: 3 / Source method: obtained synthetically / Formula: C32H59N2O8S / Comment: detergent*YM
#11: Chemical ChemComp-AF3 / ALUMINUM FLUORIDE / Aluminium fluoride


Mass: 83.977 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF3

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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 backtracked complex bound to nsp13 helicase - nsp13(1)-BTC
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2 (unknown)
Source (recombinant)Organism: Escherichia coli K-12 (unknown)
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: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 404706 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00717884
ELECTRON MICROSCOPYf_angle_d0.70324672
ELECTRON MICROSCOPYf_dihedral_angle_d15.8426748
ELECTRON MICROSCOPYf_chiral_restr0.0462876
ELECTRON MICROSCOPYf_plane_restr0.0042832

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