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- EMDB-24103: Cryo-EM structure of the monomeric form of SARS-CoV-2 nsp10-nsp14... -

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Entry
Database: EMDB / ID: EMD-24103
TitleCryo-EM structure of the monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex
Map data
Sample
  • Complex: Monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex
    • Protein or peptide: Non-structural protein 10
    • Protein or peptide: Proofreading exoribonuclease
  • RNA: RNA (25-MER)
  • RNA: RNA (24-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsSARS-CoV-2 / exoribonuclease / mismatch correction / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / ISG15-specific peptidase activity / TRAF3-dependent IRF activation pathway ...protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / ISG15-specific peptidase activity / TRAF3-dependent IRF activation pathway / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / 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 / host cell endoplasmic reticulum-Golgi intermediate compartment / SARS coronavirus main proteinase / 3'-5'-RNA exonuclease activity / 5'-3' DNA helicase activity / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / omega peptidase activity / SARS-CoV-2 modulates host translation machinery / mRNA (guanine-N7)-methyltransferase / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / symbiont-mediated suppression of host NF-kappaB cascade / DNA helicase / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / regulation of autophagy / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated suppression of host gene expression / copper ion binding / viral translational frameshifting / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / lipid binding / 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 / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface domain profile. / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Nidovirus 2-O-methyltransferase / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / 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 / Nonstructural protein 13, 1B domain, coronavirus / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 3'-5' exoribonuclease (ExoN) 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 / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / : / Lipocalin signature. / DNA2/NAM7 helicase-like, C-terminal / AAA domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / 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. / 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 / NSP1, C-terminal domain, betacoronavirus / : / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3e group 2-specific marker (G2M) 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, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Papain-like viral protease, palm and finger domains, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP2, N-terminal / : / Coronavirus replicase NSP2, C-terminal / Coronavirus (CoV) Nsp2 middle domain profile. / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Peptidase family C16 domain profile. / : / : / Coronavirus replicase NSP7 / Coronavirus 3Ecto domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile.
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
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, 2021-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7n0c
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24103.png

Downloads & links

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Map

FileDownload / File: emd_24103.map.gz / Format: CCP4 / Size: 64 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 256 pix.
= 273.408 Å		1.07 Å/pix.
x 256 pix.
= 273.408 Å		1.07 Å/pix.
x 256 pix.
= 273.408 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.068 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-4.070384 - 5.6158724
Average (Standard dev.)0.0018435735 (±0.079105295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.408 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0681.0681.068
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.408273.408273.408
α/β/γ90.00090.00090.000
start NX/NY/NZ-288-288-288
NX/NY/NZ576576576
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-4.0705.6160.002

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Supplemental data

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Sample components

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Entire : Monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex

EntireName: Monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex
Components
  • Complex: Monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex
    • Protein or peptide: Non-structural protein 10
    • Protein or peptide: Proofreading exoribonuclease
  • RNA: RNA (25-MER)
  • RNA: RNA (24-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex

SupramoleculeName: Monomeric form of SARS-CoV-2 nsp10-nsp14 (E191A)-RNA complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Macromolecule #1: Non-structural protein 10

MacromoleculeName: Non-structural protein 10 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 14.80293 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AGNATEVPAN STVLSFCAFA VDAAKAYKDY LASGGQPITN CVKMLCTHTG TGQAITVTPE ANMDQESFGG ASCCLYCRCH IDHPNPKGF CDLKGKYVQI PTTCANDPVG FTLKNTVCTV CGMWKGYGCS CDQLREPMLQ

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #2: Proofreading exoribonuclease

MacromoleculeName: Proofreading exoribonuclease / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 59.829441 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AENVTGLFKD CSKVITGLHP TQAPTHLSVD TKFKTEGLCV DIPGIPKDMT YRRLISMMGF KMNYQVNGYP NMFITREEAI RHVRAWIGF DVEGCHATRE AVGTNLPLQL GFSTGVNLVA VPTGYVDTPN NTDFSRVSAK PPPGDQFKHL IPLMYKGLPW N VVRIKIVQ ...String:
AENVTGLFKD CSKVITGLHP TQAPTHLSVD TKFKTEGLCV DIPGIPKDMT YRRLISMMGF KMNYQVNGYP NMFITREEAI RHVRAWIGF DVEGCHATRE AVGTNLPLQL GFSTGVNLVA VPTGYVDTPN NTDFSRVSAK PPPGDQFKHL IPLMYKGLPW N VVRIKIVQ MLSDTLKNLS DRVVFVLWAH GFALTSMKYF VKIGPERTCC LCDRRATCFS TASDTYACWH HSIGFDYVYN PF MIDVQQW GFTGNLQSNH DLYCQVHGNA HVASCDAIMT RCLAVHECFV KRVDWTIEYP IIGDELKINA ACRKVQHMVV KAA LLADKF PVLHDIGNPK AIKCVPQADV EWKFYDAQPC SDKAYKIEEL FYSYATHSDK FTDGVCLFWN CNVDRYPANS IVCR FDTRV LSNLNLPGCD GGSLYVNKHA FHTPAFDKSA FVNLKQLPFF YYSDSPCESH GKQVVSDIDY VPLKSATCIT RCNLG GAVC RHHANEYRLY LDAYNMMISA GFSLWVYKQF DTYNLWNTFT RLQ

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #3: RNA (25-MER)

MacromoleculeName: RNA (25-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 11.901023 KDa
SequenceString:
GGGAUGUGAU UUUAAUAGCU UCUUAGGAGA AUGACUU

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Macromolecule #4: RNA (24-MER)

MacromoleculeName: RNA (24-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 10.459259 KDa
SequenceString:
CGGUCAUUCU CCUAAGAAGC UAUUAAAAUC ACC

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 5 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.19 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5c8u

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 184500
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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