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- PDB-7krp: Structure of SARS-CoV-2 backtracked complex complex bound to nsp1... -

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Basic information

Entry
Database: PDB / ID: 7krp
TitleStructure of SARS-CoV-2 backtracked complex complex bound to nsp13 helicase - BTC (local refinement)
Components
  • (Non-structural protein ...Viral nonstructural protein) x 2
  • RNA (36-MER)
  • RNA (37-MER)
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsTRANSFERASE/RNA / RNA-dependent RNA polymerase / viral replication-transcription complex / transcription / viral proteins / TRANSFERASE-RNA complex
Function / homology
Function and homology information


Maturation of replicase proteins / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Transcription of SARS-CoV-2 sgRNAs / host cell endosome / Translation of Replicase and Assembly of the Replication Transcription Complex / suppression by virus of host TBK1 activity / exoribonuclease activity / modulation by virus of host autophagy / mRNA methylation / RNA phosphodiester bond hydrolysis, exonucleolytic ...Maturation of replicase proteins / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Transcription of SARS-CoV-2 sgRNAs / host cell endosome / Translation of Replicase and Assembly of the Replication Transcription Complex / suppression by virus of host TBK1 activity / exoribonuclease activity / modulation by virus of host autophagy / mRNA methylation / RNA phosphodiester bond hydrolysis, exonucleolytic / double membrane vesicle viral factory outer membrane / suppression by virus of host translation / ISG15-specific protease activity / host cell Golgi apparatus / Replication of the SARS-CoV-2 genome / suppression by virus of host type I interferon production / host cell endoplasmic reticulum / induction by virus of catabolism of host mRNA / SARS coronavirus main proteinase / cytoplasmic viral factory / G-quadruplex RNA binding / suppression by virus of host ISG15 activity / suppression by virus of host toll-like receptor signaling pathway / protein K48-linked deubiquitination / 3'-5'-exoribonuclease activity / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / host cell endoplasmic reticulum-Golgi intermediate compartment / suppression by virus of host IRF3 activity / suppression by virus of host NF-kappaB transcription factor activity / modulation by virus of host protein ubiquitination / transcription, RNA-templated / protein K63-linked deubiquitination / viral genome replication / positive stranded viral RNA replication / protein autoprocessing / suppression by virus of host TRAF activity / Transferases; Transferring one-carbon groups; Methyltransferases / mRNA (nucleoside-2'-O-)-methyltransferase activity / helicase activity / ubiquitinyl hydrolase 1 / viral transcription / cysteine-type peptidase activity / methyltransferase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / DNA helicase / thiol-dependent deubiquitinase / DNA helicase activity / single-stranded RNA binding / host cell perinuclear region of cytoplasm / methylation / RNA helicase / induction by virus of host autophagy / RNA-directed RNA polymerase / endonuclease activity / viral protein processing / suppression by virus of host type I interferon-mediated signaling pathway / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / RNA helicase activity / host cell cytoplasm / transcription, DNA-templated / Hydrolases; Acting on ester bonds / protein dimerization 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 / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nonstructural protein 14, betacoronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nonstructural protein 14, betacoronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, coronavirus / Non-structural protein NSP3, N-terminal, betacoronavirus / DPUP/SUD, C-terminal, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein 2, SARS-CoV-like / Non-structural protein NSP1 superfamily, betacoronavirus / Viral (Superfamily 1) RNA helicase / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / Betacoronavirus Nsp3c-M domain profile. / Nonstructural protein 13, 1B domain, coronavirus / NSP15, NendoU domain, coronavirus / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / Coronavirus replicase NSP15, N-terminal oligomerisation / Coronavirus replicase NSP15, middle domain / Non-structural protein NSP16, coronavirus-like / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, middle domain / Coronavirus 2'-O-methyltransferase / Non-structural protein 14, coronavirus / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / RNA polymerase, N-terminal, coronaviral / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP15, N-terminal oligomerisation / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Carbamoyl-phosphate synthase subdomain signature 2. / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / Endoribonuclease EndoU-like / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Betacoronavirus single-stranded poly(A) binding domain / Non-structural protein NSP1, betacoronavirus / Betacoronavirus replicase NSP1 / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Replicase polyprotein, nucleic acid-binding domain superfamily / Non-structural protein 6, betacoronavirus / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Non-structural protein NSP3, nucleic acid-binding (NAR) domain, betacoronavirus / Betacoronavirus nucleic acid-binding (NAR) / Papain-like protease, N-terminal domain superfamily, coronavirus / Papain-like protease, thumb domain superfamily, coronavirus / Non-structural protein NSP3A domain-like superfamily / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus Nsp3a Ubl domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / (+)RNA virus helicase core domain profile. / (+) RNA virus helicase core domain / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, N-terminal / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Peptidase family C16 domain profile. / Non-structural protein NSP7, coronavirus / Non-structural protein NSP7 superfamily, coronavirus / Coronavirus replicase NSP7 / Coronavirus endopeptidase C30 / Coronavirus replicase NSP4, C-terminal / Coronavirus replicase NSP3, C-terminal / Coronavirus main protease (M-pro) domain profile. / Coronavirus papain-like peptidase / Coronavirus replicase NSP8 / Non-structural protein NSP4, C-terminal, coronavirus / Coronavirus replicase NSP3, C-terminal / Coronavirus RNA synthesis protein NSP10 / Non-structural protein 6, coronavirus / Coronavirus replicase NSP4, N-terminal / Papain-like viral protease, palm and finger domains, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Non-structural protein NSP8, coronavirus-like / Non-structural protein NSP9, coronavirus / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP8 superfamily, coronavirus / Peptidase C16, coronavirus / Peptidase C30, coronavirus / Coronavirus replicase NSP4, N-terminal / Non-structural protein NSP9 superfamily, coronavirus / Peptidase C30, domain 3, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Coronavirus replicase NSP6 / Coronavirus replicase NSP9 / Lipocalin signature. / Non-structural protein 3, X-domain-like
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / RNA (> 10) / RNA / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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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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
P: RNA (37-MER)
T: RNA (36-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)193,31713
Polymers190,8396
Non-polymers2,4787
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 , 1 types, 1 molecules A

#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
Gene: rep, 1a-1b / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0DTD1, RNA-directed RNA polymerase

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

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RNA chain , 2 types, 2 molecules PT

#4: 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
#5: RNA chain RNA (36-MER)


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

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Non-polymers , 4 types, 7 molecules

#6: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#9: 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

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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 complex bound to nsp13 helicase - BTC (local refinement)
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli K-12 (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: 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: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 871163 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412978
ELECTRON MICROSCOPYf_angle_d0.5517967
ELECTRON MICROSCOPYf_dihedral_angle_d15.0974939
ELECTRON MICROSCOPYf_chiral_restr0.0392101
ELECTRON MICROSCOPYf_plane_restr0.0032007

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