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- PDB-7n0c: Cryo-EM structure of the monomeric form of SARS-CoV-2 nsp10-nsp14... -

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Basic information

Entry
Database: PDB / ID: 7n0c
TitleCryo-EM structure of the monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex
Components
  • Non-structural protein 10
  • Proofreading exoribonuclease
  • RNA (24-MER)
  • RNA (25-MER)
KeywordsVIRAL PROTEIN/RNA / SARS-CoV-2 / exoribonuclease / mismatch correction / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / viral RNA-directed RNA polymerase complex / exoribonuclease complex / endoribonuclease complex / Transcription of SARS-CoV-2 sgRNAs / endopeptidase complex / 5'-3' RNA helicase activity / mRNA cap methyltransferase complex ...Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / viral RNA-directed RNA polymerase complex / exoribonuclease 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 / modulation by virus of host autophagy / Lyases; Phosphorus-oxygen lyases / 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 / host cell endosome / 3'-5'-exoribonuclease activity / 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 / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nonstructural protein 14, betacoronavirus / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nonstructural protein 14, betacoronavirus / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) 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 / Coronavirus replicase NSP15, middle domain / Non-structural protein NSP16, coronavirus-like / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Coronavirus RNA-dependent RNA polymerase, N-terminal / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Nonstructural protein 15, middle domain, coronavirus / Non-structural protein NSP15, middle domain superfamily / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Coronaviridae zinc-binding (CV ZBD) domain profile. / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / NendoU domain, nidovirus / Endoribonuclease EndoU-like / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Non-structural protein NSP1 superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Non-structural protein 2, SARS-CoV-like / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Non-structural protein NSP3, N-terminal, betacoronavirus / Betacoronavirus replicase NSP3, N-terminal / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / 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 replicase NSP1 / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP1, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus single-stranded poly(A) binding domain / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / 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 / Coronavirus replicase NSP2, N-terminal / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus replicase NSP2, C-terminal / 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 / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Non-structural protein NSP7, coronavirus / Coronavirus RNA synthesis protein NSP10 / Non-structural protein NSP4, N-terminal, coronavirus / Coronavirus endopeptidase C30 / Non-structural protein 6, coronavirus / Coronavirus replicase NSP3, C-terminal / Coronavirus replicase NSP8 / Non-structural protein NSP8 superfamily, coronavirus / Peptidase C30, domain 3, coronavirus / Non-structural protein NSP9, coronavirus / Coronavirus main protease (M-pro) domain profile. / Coronavirus replicase NSP3, C-terminal / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Coronavirus replicase NSP6 / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Coronavirus replicase NSP4, N-terminal / Non-structural protein NSP8, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP7 superfamily, coronavirus
Similarity search - Domain/homology
RNA (> 10) / RNA / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLiu, C. / Yang, Y.
CitationJournal: Science / Year: 2021
Title: Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme.
Authors: Chang Liu / Wei Shi / Scott T Becker / David G Schatz / Bin Liu / Yang Yang /
Abstract: Coronavirus 3′-to-5′ exoribonuclease (ExoN), residing in the nonstructural protein (nsp) 10–nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the ...Coronavirus 3′-to-5′ exoribonuclease (ExoN), residing in the nonstructural protein (nsp) 10–nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the virus life cycle. ExoN also recognizes and excises nucleotide analog inhibitors incorporated into the nascent RNA, undermining the effectiveness of nucleotide analog–based antivirals. Here we present cryo–electron microscopy structures of both wild-type and mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp10-nsp14 in complex with an RNA substrate bearing a 3′-end mismatch at resolutions ranging from 2.5 to 3.9 angstroms. The structures reveal the molecular determinants of ExoN substrate specificity and offer insight into the molecular mechanisms of mismatch correction during coronavirus RNA synthesis. Our findings provide guidance for rational design of improved anticoronavirus therapies.
History
DepositionMay 25, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 4, 2021Group: Database references / Category: citation / Item: _citation.pdbx_database_id_DOI
Revision 1.2Aug 25, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Sep 22, 2021Group: 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: Non-structural protein 10
B: Proofreading exoribonuclease
T: RNA (25-MER)
D: RNA (24-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,36811
Polymers96,9934
Non-polymers3767
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8850 Å2
ΔGint-64 kcal/mol
Surface area35900 Å2

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Non-structural protein 10 / nsp10 / Growth factor-like peptide / GFL


Mass: 14802.930 Da / Num. of mol.: 1 / Fragment: UNP residues 4254-4392
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 (E. coli) / References: UniProt: P0DTD1
#2: Protein Proofreading exoribonuclease / ExoN / Guanine-N7 methyltransferase / Non-structural protein 14 / nsp14


Mass: 59829.441 Da / Num. of mol.: 1 / Fragment: UNP residues 5926-6452 / Mutation: E191A
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 (E. coli)
References: UniProt: P0DTD1, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters

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RNA chain , 2 types, 2 molecules TD

#3: RNA chain RNA (25-MER)


Mass: 11901.023 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2
#4: RNA chain RNA (24-MER)


Mass: 10459.259 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2

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Non-polymers , 3 types, 8 molecules

#5: Chemical
ChemComp-ZN / ZINC ION


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


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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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: Monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: 49.19 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184500 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0086339
ELECTRON MICROSCOPYf_angle_d0.7878841
ELECTRON MICROSCOPYf_dihedral_angle_d7.2923624
ELECTRON MICROSCOPYf_chiral_restr0.0471015
ELECTRON MICROSCOPYf_plane_restr0.006953

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