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- PDB-7bv1: Cryo-EM structure of the apo nsp12-nsp7-nsp8 complex -

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Basic information

Entry
Database: PDB / ID: 7bv1
TitleCryo-EM structure of the apo nsp12-nsp7-nsp8 complex
Components
  • Non-structural protein 7
  • Non-structural protein 8
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN / SARS-CoV-2 / RNA Polymerase / Apo
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs ...protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / TRAF3-dependent IRF activation pathway / snRNP Assembly / Replication of the SARS-CoV-2 genome / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / SARS coronavirus main proteinase / host cell endosome / 3'-5'-RNA exonuclease activity / : / host cell endoplasmic reticulum-Golgi intermediate compartment / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / SARS-CoV-2 modulates host translation machinery / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / DNA helicase / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell endoplasmic reticulum membrane / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / RNA helicase / induction by virus of host autophagy / RNA-directed RNA polymerase / copper ion binding / symbiont-mediated suppression of host gene expression / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / lipid binding / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / : / Coronavirus Nsp12 Interface domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / : / Coronavirus Nsp12 Interface domain profile. / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / NSP12 RNA-dependent RNA polymerase, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus 2-O-methyltransferase / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nonstructural protein 13, 1B domain, coronavirus / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / 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 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 / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / DNA2/NAM7 helicase-like, C-terminal / AAA domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Lipocalin signature. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / NSP1 globular domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Carbamoyl-phosphate synthase subdomain signature 2. / Coronavirus 3Ecto domain profile. / : / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / NSP1, C-terminal domain, betacoronavirus / Betacoronavirus Nsp3c-M domain profile. / 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 / : / Coronavirus (CoV) Nsp2 middle domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / NSP1, globular domain, alpha/betacoronavirus / : / Coronavirus (CoV) Nsp3 Y domain profile. / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus replicase NSP2, N-terminal / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Non-structural protein 2, C-terminal domain, coronavirus / 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
Similarity search - Domain/homology
Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYin, W. / Mao, C. / Luan, X. / Shen, D. / Shen, Q. / Su, H. / Wang, X. / Zhou, F. / Zhao, W. / Gao, M. ...Yin, W. / Mao, C. / Luan, X. / Shen, D. / Shen, Q. / Su, H. / Wang, X. / Zhou, F. / Zhao, W. / Gao, M. / Chang, S. / Xie, Y.C. / Tian, G. / Jiang, H.W. / Tao, S.C. / Shen, J. / Jiang, Y. / Jiang, H. / Xu, Y. / Zhang, S. / Zhang, Y. / Xu, H.E.
CitationJournal: Science / Year: 2020
Title: Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir.
Authors: Wanchao Yin / Chunyou Mao / Xiaodong Luan / Dan-Dan Shen / Qingya Shen / Haixia Su / Xiaoxi Wang / Fulai Zhou / Wenfeng Zhao / Minqi Gao / Shenghai Chang / Yuan-Chao Xie / Guanghui Tian / He- ...Authors: Wanchao Yin / Chunyou Mao / Xiaodong Luan / Dan-Dan Shen / Qingya Shen / Haixia Su / Xiaoxi Wang / Fulai Zhou / Wenfeng Zhao / Minqi Gao / Shenghai Chang / Yuan-Chao Xie / Guanghui Tian / He-Wei Jiang / Sheng-Ce Tao / Jingshan Shen / Yi Jiang / Hualiang Jiang / Yechun Xu / Shuyang Zhang / Yan Zhang / H Eric Xu /
Abstract: The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral ...The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.
History
DepositionApr 9, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 22, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Source and taxonomy / Structure summary
Category: citation / entity ...citation / entity / entity_name_com / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _entity.pdbx_description / _entity.pdbx_ec / _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_gene_src_gene / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq.seq_align_beg / _struct_ref_seq.seq_align_end
Revision 2.0May 27, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: atom_site / citation ...atom_site / citation / citation_author / em_software / pdbx_poly_seq_scheme / pdbx_struct_assembly_prop / pdbx_struct_conn_angle / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / refine / refine_ls_restr / software / struct_conf / struct_conn / struct_mon_prot_cis / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID ..._citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name / _em_software.category / _em_software.fitting_id / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_struct_assembly_prop.value / _pdbx_struct_conn_angle.value / _refine.B_iso_mean / _refine_ls_restr.dev_ideal / _refine_ls_restr.number / _software.version / _struct_conn.pdbx_dist_value / _struct_sheet.number_strands
Description: Sequence discrepancy
Details: A sequence misalignment by 9 residues in the C-terminal fragment inherited from 6nur and 7btf.
Provider: author / Type: Coordinate replacement
Revision 2.1Jun 10, 2020Group: Structure summary / Category: entity / struct / Item: _entity.pdbx_ec / _struct.pdbx_descriptor
Revision 2.2Jul 15, 2020Group: 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
Revision 2.3Mar 10, 2021Group: Structure summary / Category: entity / entity_name_com / Item: _entity.pdbx_description / _entity_name_com.name
Revision 2.4Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,9656
Polymers165,8344
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8620 Å2
ΔGint-84 kcal/mol
Surface area46570 Å2

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Components

#1: Protein RNA-directed RNA polymerase / RNA-dependent RNA polymerase / pp1ab / ORF1ab polyprotein / Pol / RdRp / Non-structural protein 12 / nsp12


Mass: 109070.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0DTD1, RNA-directed RNA polymerase
#2: Protein Non-structural protein 8 / pp1ab / ORF1ab polyprotein / nsp8


Mass: 23139.418 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0DTD1
#3: Protein Non-structural protein 7 / pp1ab / ORF1ab polyprotein / nsp7


Mass: 10485.176 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0DTD1
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
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

ComponentName: the apo nsp12-nsp7-nsp8 complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: SARS bat coronavirus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
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: 64 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18_3845refinement
PHENIX1.18_3845refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81494 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 30.86 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058992
ELECTRON MICROSCOPYf_angle_d0.818112222
ELECTRON MICROSCOPYf_chiral_restr0.05831393
ELECTRON MICROSCOPYf_plane_restr0.00471555
ELECTRON MICROSCOPYf_dihedral_angle_d18.78473209

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