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- PDB-6xez: Structure of SARS-CoV-2 replication-transcription complex bound t... -

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Entry
Database: PDB / ID: 6xez
TitleStructure of SARS-CoV-2 replication-transcription complex bound to nsp13 helicase - nsp13(2)-RTC
Components
  • Product RNA
  • Template RNA
  • nsp12
  • nsp13
  • nsp7
  • nsp8
KeywordsTRANSFERASE/HYDROLASE/RNA / RNA-dependent RNA polymerase / viral replication-transcription complex / transcription / viral proteins / TRANSFERASE-HYDROLASE-RNA complex
Function / homology
Function and homology information


SARS coronavirus main proteinase / induction by virus of catabolism of host mRNA / suppression by virus of host ISG15 activity / exonuclease activity / host cell endoplasmic reticulum-Golgi intermediate compartment / suppression by virus of host NF-kappaB transcription factor activity / modulation by virus of host protein ubiquitination / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / go:0036459: / ubiquitinyl hydrolase 1 ...SARS coronavirus main proteinase / induction by virus of catabolism of host mRNA / suppression by virus of host ISG15 activity / exonuclease activity / host cell endoplasmic reticulum-Golgi intermediate compartment / suppression by virus of host NF-kappaB transcription factor activity / modulation by virus of host protein ubiquitination / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / go:0036459: / ubiquitinyl hydrolase 1 / Transferases; Transferring one-carbon groups; Methyltransferases / DNA helicase / DNA helicase activity / methyltransferase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / host cell perinuclear region of cytoplasm / host cell membrane / suppression by virus of host type I interferon-mediated signaling pathway / RNA helicase / methylation / induction by virus of host autophagy / RNA-directed RNA polymerase / endonuclease activity / RNA helicase activity / RNA-directed 5'-3' RNA polymerase activity / Hydrolases; Acting on ester bonds / RNA binding / integral component of membrane / ATP binding / metal ion binding
Peptidase S1, PA clan / Non-structural protein NSP15, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus / S-adenosyl-L-methionine-dependent methyltransferase / Macro domain / Peptidase C30, coronavirus / P-loop containing nucleoside triphosphate hydrolase / Non-structural protein NSP16, coronavirus-like / RNA polymerase, N-terminal, coronaviral / RNA synthesis protein NSP10 superfamily, coronavirus ...Peptidase S1, PA clan / Non-structural protein NSP15, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus / S-adenosyl-L-methionine-dependent methyltransferase / Macro domain / Peptidase C30, coronavirus / P-loop containing nucleoside triphosphate hydrolase / Non-structural protein NSP16, coronavirus-like / RNA polymerase, N-terminal, coronaviral / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP9, coronavirus / in:ipr014827: / Non-structural protein NSP7, coronavirus / Non-structural protein NSP8, coronavirus-like / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP1, betacoronavirus / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, single-stranded poly(A) binding domain, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding (NAR) domain, betacoronavirus / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Papain-like protease, thumb domain superfamily, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Replicase polyprotein, nucleic acid-binding domain superfamily / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, coronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP1 superfamily, betacoronavirus / Non-structural protein NSP8 superfamily, coronavirus / Endoribonuclease EndoU-like / Non-structural protein NSP7 superfamily, coronavirus / Non-structural protein NSP3, N-terminal, betacoronavirus
Replicase polyprotein 1ab
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsChen, J. / Malone, B. / Llewellyn, E.C. / Campbell, E.A. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
Citation
Journal: bioRxiv / Year: 2020
Title: Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex.
Authors: James Chen / Brandon Malone / Eliza Llewellyn / Michael Grasso / Patrick M M Shelton / Paul Dominic B Olinares / Kashyap Maruthi / Edward T Eng / Hasan Vatandaslar / Brian Chait / Tarun ...Authors: James Chen / Brandon Malone / Eliza Llewellyn / Michael Grasso / Patrick M M Shelton / Paul Dominic B Olinares / Kashyap Maruthi / Edward T Eng / Hasan Vatandaslar / Brian Chait / Tarun Kapoor / Seth A Darst / Elizabeth A Campbell
Abstract: SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a ...SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.
#1: Journal: bioRxiv / Year: 2020
Title: Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex.
Authors: James Chen / Brandon Malone / Eliza Llewellyn / Michael Grasso / Patrick M M Shelton / Paul Dominic B Olinares / Kashyap Maruthi / Edward T Eng / Hasan Vatandaslar / Brian Chait / Tarun ...Authors: James Chen / Brandon Malone / Eliza Llewellyn / Michael Grasso / Patrick M M Shelton / Paul Dominic B Olinares / Kashyap Maruthi / Edward T Eng / Hasan Vatandaslar / Brian Chait / Tarun Kapoor / Seth A Darst / Elizabeth A Campbell
Abstract: SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a ...SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Category: citation / citation_author

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Structure visualization

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Assembly

Deposited unit
A: nsp12
B: nsp8
C: nsp7
D: nsp8
E: nsp13
F: nsp13
P: Product RNA
T: Template RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)327,96227
Polymers324,0218
Non-polymers3,94119
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24560 Å2
ΔGint-236 kcal/mol
Surface area125760 Å2

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Components

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Protein , 4 types, 6 molecules ABDCEF

#1: Protein nsp12 / RNA-directed RNA polymerase


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 BL21(DE3) (bacteria)
References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA ...References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA helicase, RNA helicase, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds, Transferases; Transferring one-carbon groups; Methyltransferases
#2: Protein 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 BL21(DE3) (bacteria)
References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA ...References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA helicase, RNA helicase, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds, Transferases; Transferring one-carbon groups; Methyltransferases
#3: Protein 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 BL21(DE3) (bacteria)
References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA ...References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA helicase, RNA helicase, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds, Transferases; Transferring one-carbon groups; Methyltransferases
#4: Protein nsp13 / helicase


Mass: 67354.039 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
Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA ...References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA helicase, RNA helicase, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds, Transferases; Transferring one-carbon groups; Methyltransferases

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RNA chain , 2 types, 2 molecules PT

#5: RNA chain Product RNA


Mass: 11141.644 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2
#6: RNA chain Template RNA


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 , 5 types, 19 molecules

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


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


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / 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.: 2 / Source method: obtained synthetically / Formula: AlF3 / 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

ComponentName: SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - nsp13(2)-RTC
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6 / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58942 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0122357
ELECTRON MICROSCOPYf_angle_d0.94530746
ELECTRON MICROSCOPYf_dihedral_angle_d12.3763689
ELECTRON MICROSCOPYf_chiral_restr0.0523574
ELECTRON MICROSCOPYf_plane_restr0.0053643

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