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- PDB-7eiz: Coupling of N7-methyltransferase and 3'-5' exoribonuclease with S... -

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Entry
Database: PDB / ID: 7eiz
TitleCoupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading
Components
  • (Non-structural protein ...Viral nonstructural protein) x 4
  • Helicase
  • Proofreading exoribonuclease
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
  • primer
  • template
KeywordsVIRAL PROTEIN/RNA / VIRAL PROTEIN-RNA COMPLEX
Function / homology
Function and homology information


Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Transcription of SARS-CoV-2 sgRNAs / host cell endosome / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Translation of Replicase and Assembly of the Replication Transcription Complex / exoribonuclease activity / RNA phosphodiester bond hydrolysis, exonucleolytic / modulation by virus of host autophagy / mRNA methylation ...Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Transcription of SARS-CoV-2 sgRNAs / host cell endosome / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Translation of Replicase and Assembly of the Replication Transcription Complex / exoribonuclease activity / RNA phosphodiester bond hydrolysis, exonucleolytic / modulation by virus of host autophagy / mRNA methylation / 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 / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / G-quadruplex RNA binding / 3'-5'-exoribonuclease activity / suppression by virus of host ISG15-protein conjugation / host cell endoplasmic reticulum-Golgi intermediate compartment / protein K48-linked deubiquitination / suppression by virus of host toll-like receptor signaling pathway / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / transcription, RNA-templated / suppression by virus of host NF-kappaB cascade / modulation by virus of host protein ubiquitination / protein K63-linked deubiquitination / positive stranded viral RNA replication / protein autoprocessing / cysteine-type peptidase activity / mRNA (nucleoside-2'-O-)-methyltransferase activity / viral genome replication / suppression by virus of host TRAF activity / helicase activity / viral transcription / Transferases; Transferring one-carbon groups; Methyltransferases / ubiquitinyl hydrolase 1 / methyltransferase activity / DNA helicase / thiol-dependent deubiquitinase / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / DNA helicase activity / single-stranded RNA binding / host cell perinuclear region of cytoplasm / transferase activity / methylation / RNA helicase / induction by virus of host autophagy / endonuclease activity / RNA-directed RNA polymerase / RNA helicase activity / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / viral protein processing / suppression by virus of host type I interferon-mediated signaling pathway / Hydrolases; Acting on ester bonds / transcription, DNA-templated / host cell cytoplasm / 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 / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nonstructural protein 14, betacoronavirus / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nonstructural protein 14, betacoronavirus / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) 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 NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / Coronavirus replicase NSP15, middle domain / Non-structural protein NSP15, middle domain superfamily / Coronavirus 2'-O-methyltransferase / Coronavirus replicase NSP15, N-terminal oligomerisation / Coronavirus replicase NSP15, N-terminal oligomerisation / Coronavirus replicase NSP15, middle domain / RNA polymerase, N-terminal, coronavirus / Coronavirus RNA-dependent RNA polymerase, N-terminal / Coronavirus proofreading exoribonuclease / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Non-structural protein NSP1 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 / Polyprotein cleavage domain PL2pro superfamily, coronavirus / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / Endoribonuclease EndoU-like / Carbamoyl-phosphate synthase subdomain signature 2. / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Betacoronavirus Nsp3c-M domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / Betacoronavirus Nsp3c-C domain profile. / (+)RNA virus helicase core domain profile. / (+) RNA virus helicase core domain / Non-structural protein NSP1, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Betacoronavirus single-stranded poly(A) binding domain / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Replicase polyprotein, nucleic acid-binding domain superfamily / Coronavirus (CoV) Nsp1 globular domain profile. / Non-structural protein 6, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3A domain-like superfamily / Papain-like protease, N-terminal domain superfamily, coronavirus / 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. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Papain-like protease, thumb domain superfamily, coronavirus / Lipocalin signature. / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus Nsp3a Ubl domain profile. / Coronavirus replicase NSP2, N-terminal / Nonstructural protein 2, N-terminal domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Coronavirus replicase NSP7 / Non-structural protein NSP7, coronavirus / Non-structural protein NSP7 superfamily, coronavirus / Peptidase family C16 domain profile. / Peptidase C16, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus / Coronavirus main protease (M-pro) domain profile. / Coronavirus RNA synthesis protein NSP10 / Coronavirus replicase NSP8 / Peptidase C30, coronavirus / Coronavirus papain-like peptidase / Coronavirus endopeptidase C30 / Coronavirus replicase NSP4, N-terminal / Non-structural protein NSP9, coronavirus / Non-structural protein NSP8, coronavirus-like / RNA synthesis protein NSP10, coronavirus / Coronavirus replicase NSP4, C-terminal / Coronavirus replicase NSP6 / Non-structural protein 6, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP8 superfamily, coronavirus / Peptidase C30, domain 3, coronavirus / Coronavirus replicase NSP4, N-terminal
Similarity search - Domain/homology
RNA / RNA (> 10) / Replicase polyprotein 1a / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM
AuthorsYan, L. / Yang, Y.X. / Li, M.Y. / Zhang, Y. / Zheng, L.T. / Ge, J. / Huang, Y.C. / Liu, Z.Y. / Wang, T. / Gao, S. ...Yan, L. / Yang, Y.X. / Li, M.Y. / Zhang, Y. / Zheng, L.T. / Ge, J. / Huang, Y.C. / Liu, Z.Y. / Wang, T. / Gao, S. / Zhang, R. / Huang, Y.Y. / Guddat, L.W. / Gao, Y. / Rao, Z.H. / Lou, Z.Y.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2017YFC0840300 China
Ministry of Science and Technology (MoST, China)2020YFA0707500 China
CitationJournal: Cell / Year: 2021
Title: Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.
Authors: Liming Yan / Yunxiang Yang / Mingyu Li / Ying Zhang / Litao Zheng / Ji Ge / Yucen C Huang / Zhenyu Liu / Tao Wang / Shan Gao / Ran Zhang / Yuanyun Y Huang / Luke W Guddat / Yan Gao / Zihe Rao / Zhiyong Lou /
Abstract: The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex ...The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (GpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.
History
DepositionApr 1, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Database references / Category: citation / Item: _citation.title

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Structure visualization

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Structure viewerMolecule:
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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
G: Non-structural protein 9
H: Non-structural protein 10
I: primer
J: template
K: Proofreading exoribonuclease
E: Helicase
F: Helicase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)398,00825
Polymers397,13311
Non-polymers87514
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27240 Å2
ΔGint-209 kcal/mol
Surface area153320 Å2

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Components

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Protein , 3 types, 4 molecules AKEF

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


Mass: 106440.562 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) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1, RNA-directed RNA polymerase
#8: Protein Proofreading exoribonuclease / ExoN / Guanine-N7 methyltransferase / Non-structural protein 14 / nsp14


Mass: 59887.480 Da / Num. of mol.: 1 / Fragment: UNP residues 5926-6452
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: P0DTD1, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
#9: Protein Helicase / / Hel / Non-structural protein 13 / nsp13


Mass: 66930.531 Da / Num. of mol.: 2 / Fragment: UNP residues 5325-5925
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1

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Non-structural protein ... , 4 types, 5 molecules BDCGH

#2: Protein Non-structural protein 8 / nsp8


Mass: 21903.047 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) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1
#3: Protein Non-structural protein 7 / nsp7


Mass: 9248.804 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
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0DTC1
#4: Protein Non-structural protein 9 / nsp9


Mass: 12391.171 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1
#5: 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
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1

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RNA chain , 2 types, 2 molecules IJ

#6: RNA chain primer


Mass: 8172.932 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
#7: RNA chain template


Mass: 8522.079 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)

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Non-polymers , 2 types, 14 molecules

#10: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1mCap(0)-RTCCOMPLEX#1-#90RECOMBINANT
2NSP 7, 8, 9, 10, 12, 13, 14COMPLEX#1-#6, #91RECOMBINANT
3primer RNA, template RNACOMPLEX#7-#81RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Severe acute respiratory syndrome coronavirus 22697049
32Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
32Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
Buffer solutionpH: 7
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: 60 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18rc3_3805: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionNum. of particles: 80256 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01327791
ELECTRON MICROSCOPYf_angle_d1.0638013
ELECTRON MICROSCOPYf_dihedral_angle_d14.3064236
ELECTRON MICROSCOPYf_chiral_restr0.0554347
ELECTRON MICROSCOPYf_plane_restr0.0074671

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