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- PDB-8aou: Solution NMR structure of full-length Nsp1 from SARS-CoV-2. -

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Basic information

Entry
Database: PDB / ID: 8aou
TitleSolution NMR structure of full-length Nsp1 from SARS-CoV-2.
ComponentsHost translation inhibitor nsp1
KeywordsVIRAL PROTEIN / Non-structural protein Host translation inhibitor
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
MethodSOLUTION NMR / simulated annealing / torsion angle dynamics / molecular dynamics
AuthorsWang, Y. / Kirkpatrick, J.P. / Carlomagno, T.
Funding support1items
OrganizationGrant numberCountry
Not funded
Citation
Journal: Structure / Year: 2023
Title: Structural insights into the activity regulation of full-length non-structural protein 1 from SARS-CoV-2.
Authors: Wang, Y. / Kirkpatrick, J. / Lage, S.Z. / Carlomagno, T.
#1: Journal: Biomol NMR Assign / Year: 2021
Title: 1H, 13C and 15N backbone chemical-shift assignments of SARS-CoV-2 non-structural protein 1 (leader protein)
Authors: Wang, Y. / Kirkpatrick, J. / zur Lage, S. / Korn, S. / Neissner, K. / Schwalbe, H. / Schlundt, A. / Carlomagno, T.
History
DepositionAug 8, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 25, 2023Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.3Feb 15, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Host translation inhibitor nsp1


Theoretical massNumber of molelcules
Total (without water)19,9291
Polymers19,9291
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: NMR relaxation study
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 100structures with the lowest energy
RepresentativeModel #1closest to the average

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Components

#1: Protein Host translation inhibitor nsp1 / Leader protein / Non-structural protein 1 / nsp1


Mass: 19929.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The first two residues (GA) in the protein construct used are cloning artifacts. Therefore, the appropriate residue numbering has the first residue (G) designated as residue-number '-1', so ...Details: The first two residues (GA) in the protein construct used are cloning artifacts. Therefore, the appropriate residue numbering has the first residue (G) designated as residue-number '-1', so that the third residue has residue-number '1'.
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Plasmid: pETM11 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0DTD1

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N HSQC
121isotropic22D 1H-15N HSQC
131isotropic13D HNCO
141isotropic23D HNCO
151isotropic23D HNCA
161isotropic23D HN(CA)CB
171isotropic13D HN(CO)CA
181isotropic13D HN(COCA)CB
191isotropic13D HN(CA)CO
1101isotropic23D (H)CCH-TOCSY
1111isotropic13D H(CCO)NH
1121isotropic13D HBHA(CO)NH
1131isotropic13D HA(CO)NH
1141isotropic13D HC(C)H-TOCSY (aromatics)
1151isotropic13D (H)CCH-TOCSY (aromatics)
1161isotropic12D (HB)CB(CGCD)HD
1171isotropic12D (HB)CB(CGCDCE)HE
1181isotropic12D 1H-13C HSQC
1191isotropic22D 1H-13C HSQC
1201isotropic22D CT 1H-13C HSQC (aliphatics)
1211isotropic22D CT 1H-13C HSQC (aromatics)
1222isotropic22D CT 1H-13C HSQC (methyls)
1231isotropic23D NOESY-15N HSQC
1241isotropic23D NOESY-13C HSQC
1252isotropic2CLEANEX
1262isotropic22D SCT 1H-15N HSQC
1272isotropic22D 1H-15N TROSY-HSQC
1282isotropic22D IPAP 1H-15N HSQC
1293anisotropic22D SCT 1H-15N HSQC
1303anisotropic22D 1H-15N TROSY-HSQC
1313anisotropic22D IPAP 1H-15N HSQC

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1600 uM [U-13C; U-15N] Nsp1, 50 mM sodium phosphate, 200 mM sodium chloride, 2 mM DTT, 2 mM EDTA, 0.01 % w/v sodium azide, 10 % v/v [U-2H] D2O, 90% H2O/10% D2OMain sample used for assignment and collection of distance restraints.13C15N_sample90% H2O/10% D2O
solution2500 uM [U-10% 13C; U-100% 15N] Nsp1, 50 mM sodium phosphate, 200 mM sodium chloride, 2 mM DTT, 2 mM EDTA, 0.01 % w/v sodium azide, 10 % v/v [U-2H] D2O, 90% H2O/10% D2OSample used for stereospecific assignment of Leu and Val methyl groups, CLEANEX, and measurement of isotropic N-H splittings for RDCs.10%-13C_U-15N_sample90% H2O/10% D2O
filamentous virus3500 uM [U-10% 13C; U-100% 15N] Nsp1, 50 mM sodium phosphate, 200 mM sodium chloride, 2 mM DTT, 2 mM EDTA, 0.01 % w/w sodium azide, 10 % v/v [U-2H] D2O, 12 mg/mL Pf1 phage, 90% H2O/10% D2OMeasurement of anisotropic N-H splittings for RDCs.10%-13C_U-15N_aligned_sample90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
600 uMNsp1[U-13C; U-15N]1
50 mMsodium phosphatenatural abundance1
200 mMsodium chloridenatural abundance1
2 mMDTTnatural abundance1
2 mMEDTAnatural abundance1
0.01 % w/vsodium azidenatural abundance1
10 % v/vD2O[U-2H]1
500 uMNsp1[U-10% 13C; U-100% 15N]2
50 mMsodium phosphatenatural abundance2
200 mMsodium chloridenatural abundance2
2 mMDTTnatural abundance2
2 mMEDTAnatural abundance2
0.01 % w/vsodium azidenatural abundance2
10 % v/vD2O[U-2H]2
500 uMNsp1[U-10% 13C; U-100% 15N]3
50 mMsodium phosphatenatural abundance3
200 mMsodium chloridenatural abundance3
2 mMDTTnatural abundance3
2 mMEDTAnatural abundance3
0.01 % w/wsodium azidenatural abundance3
10 % v/vD2O[U-2H]3
12 mg/mLPf1 phagenatural abundance3
Sample conditionsIonic strength: 560 mM / Ionic strength err: 20 / Label: conditions_1 / pH: 6.5 / PH err: 0.1 / Pressure: 1 atm / Temperature: 298 K / Temperature err: 0.2

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE III HDBrukerAVANCE III HD6001Equipped with N2-cooled cryogenic inverse HCN probehead
Bruker AVANCE III HDBrukerAVANCE III HD8502Equpped with He-cooled cryogenic inverse HCN probehead

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin3.2Bruker Biospincollection
NMRPipe10.1Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
CcpNmr Analysis2.4CCPNchemical shift assignment
CcpNmr Analysis2.4CCPNpeak picking
CcpNmr Analysis2.4CCPNdata analysis
ARIA2.3Linge, O'Donoghue and Nilgesstructure calculation
CNS1.21Brunger, Adams, Clore, Gros, Nilges and Readstructure calculation
Refinement
MethodSoftware ordinal
simulated annealing1
torsion angle dynamics2
molecular dynamics3
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 10

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