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- EMDB-23007: Structure of SARS-CoV-2 backtracked complex bound to nsp13 helica... -

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Basic information

Entry
Database: EMDB / ID: EMD-23007
TitleStructure of SARS-CoV-2 backtracked complex bound to nsp13 helicase - nsp13(1)-BTC
Map data
SampleSARS-CoV-2 backtracked complex bound to nsp13 helicase - nsp13(1)-BTC
  • RNA-directed RNA polymeraseRNA-dependent RNA polymerase
  • (Non-structural protein ...Viral nonstructural protein) x 2
  • Helicase
  • (nucleic-acidNucleic acid) x 2
  • (ligand) x 5
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 / modulation by virus of host protein ubiquitination / suppression by virus of host NF-kappaB transcription factor activity / 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 / Nonstructural protein 14, betacoronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) 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. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Betacoronavirus SUD-C domain ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 14, betacoronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) 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. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Betacoronavirus SUD-C domain / Sarbecovirus Nsp3c-N domain profile. / Polyprotein cleavage domain PL2pro superfamily, coronavirus / Non-structural protein NSP1 superfamily, betacoronavirus / Non-structural protein NSP3, N-terminal, betacoronavirus / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Betacoronavirus replicase NSP3, N-terminal / 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 Nsp3e nucleic acid-binding (NAB) domain profile. / Betacoronavirus Nsp3c-C domain profile. / RNA polymerase, N-terminal, coronaviral / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Non-structural protein NSP16, coronavirus-like / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Coronavirus replicase NSP15, middle domain / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Coronavirus replicase NSP15, N-terminal oligomerisation / Non-structural protein 14, coronavirus / Coronaviridae zinc-binding (CV ZBD) domain profile. / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Carbamoyl-phosphate synthase subdomain signature 2. / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / NendoU domain, nidovirus / Endoribonuclease EndoU-like / Betacoronavirus replicase NSP1 / Replicase polyprotein, nucleic acid-binding domain superfamily / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP1, betacoronavirus / Betacoronavirus single-stranded poly(A) binding domain / Non-structural protein 6, betacoronavirus / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Betacoronavirus nucleic acid-binding (NAR) / Non-structural protein NSP3, nucleic acid-binding (NAR) domain, betacoronavirus / Papain-like protease, thumb domain superfamily, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Non-structural protein NSP3A domain-like superfamily / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / 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 / Non-structural protein NSP7 superfamily, coronavirus / Non-structural protein NSP7, coronavirus / Peptidase family C16 domain profile. / Coronavirus replicase NSP7 / Coronavirus papain-like peptidase / Coronavirus endopeptidase C30 / Papain-like viral protease, palm and finger domains, coronavirus / Coronavirus replicase NSP4, N-terminal / Coronavirus replicase NSP3, C-terminal / Non-structural protein 6, coronavirus / Coronavirus replicase NSP8 / Coronavirus RNA synthesis protein NSP10 / Peptidase C30, domain 3, coronavirus / Coronavirus replicase NSP4, C-terminal / Non-structural protein NSP9 superfamily, coronavirus / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP8, coronavirus-like / RNA synthesis protein NSP10, coronavirus / Coronavirus replicase NSP6 / Non-structural protein NSP4, C-terminal, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Coronavirus main protease (M-pro) domain profile. / Coronavirus replicase NSP4, N-terminal / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Coronavirus replicase NSP3, C-terminal / Non-structural protein NSP8 superfamily, coronavirus / Coronavirus replicase NSP9 / Lipocalin signature. / Non-structural protein 3, X-domain-like
Similarity search - Domain/homology
Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsChen J / Malone B / Campbell EA / Darst SA
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseApr 21, 2021-
Map releaseApr 21, 2021-
UpdateMay 5, 2021-
Current statusMay 5, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
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  • Surface view with fitted model
  • Atomic models: PDB-7krn
  • Surface level: 0.5
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23007.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 320 pix.
= 340.8 Å
1.07 Å/pix.
x 320 pix.
= 340.8 Å
1.07 Å/pix.
x 320 pix.
= 340.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.5
Minimum - Maximum-4.1751156 - 6.324998
Average (Standard dev.)0.0020568103 (±0.10959497)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 340.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0651.0651.065
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z340.800340.800340.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-4.1756.3250.002

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Supplemental data

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Additional map: #1

Fileemd_23007_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire SARS-CoV-2 backtracked complex bound to nsp13 helicase - nsp13(1)-BTC

EntireName: SARS-CoV-2 backtracked complex bound to nsp13 helicase - nsp13(1)-BTC
Number of components: 12

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Component #1: protein, SARS-CoV-2 backtracked complex bound to nsp13 helicase -...

ProteinName: SARS-CoV-2 backtracked complex bound to nsp13 helicase - nsp13(1)-BTC
Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #2: protein, RNA-directed RNA polymerase

ProteinName: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 106.780977 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #3: protein, Non-structural protein 8

ProteinName: Non-structural protein 8 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 22.034242 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #4: protein, Non-structural protein 7

ProteinName: Non-structural protein 7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.748385 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #5: protein, Helicase

ProteinName: Helicase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 67.354039 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #6: nucleic-acid, RNA (37-MER)

nucleic acidName: RNA (37-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CGCGUAGCAU GCUACGUCAU UCUCCUAAGA AGCUACCCCC
MassTheoretical: 12.667554 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2

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Component #7: nucleic-acid, RNA (43-MER)

nucleic acidName: RNA (43-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CUAUCCCCAU GUGAUUUUAA UAGCUUCUUA GGAGAAUGAC GUAGCAUGCU ACGCG
MassTheoretical: 17.573391 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2

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Component #8: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #9: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #10: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Component #11: ligand, CHAPSO

LigandName: CHAPSOCHAPS detergent / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.631884 kDa

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Component #12: ligand, ALUMINUM FLUORIDE

LigandName: ALUMINUM FLUORIDEAluminium fluoride / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.397705 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 66 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 404706
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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