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- PDB-7n06: SARS-CoV-2 Nsp15 endoribonuclease post-cleavage state -

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Basic information

Entry
Database: PDB / ID: 7n06
TitleSARS-CoV-2 Nsp15 endoribonuclease post-cleavage state
Components
  • RNA (5'-R(*AP*UP*A)-3')
  • Uridylate-specific endoribonuclease
KeywordsVIRAL PROTEIN / Hydrolase/RNA / endoribonuclease / Hydrolase-RNA complex
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / 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 / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / TRAF3-dependent IRF activation pathway / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-2 genome / snRNP Assembly / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / SARS coronavirus main proteinase / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / 5'-3' DNA helicase activity / symbiont-mediated suppression of host NF-kappaB cascade / host cell endosome / 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 / mRNA (guanine-N7)-methyltransferase / omega peptidase activity / methyltransferase cap1 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / SARS-CoV-2 modulates host translation machinery / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / host cell perinuclear region of cytoplasm / single-stranded RNA binding / host cell endoplasmic reticulum membrane / viral protein processing / lyase activity / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / copper ion binding / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / lipid binding / DNA-templated transcription / 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 Nonstructural protein 13, 1B domain ...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 Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Coronavirus Nsp12 Interface domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) 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 / Nidovirus 2-O-methyltransferase / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Nidovirus 3'-5' exoribonuclease domain / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / 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 / 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 / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / 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 / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp2 middle domain profile. / 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 / : / Coronavirus 3Ecto domain profile. / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / : / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Coronavirus (CoV) Nsp3 Y domain profile. / Coronavirus replicase NSP7 / Peptidase family C16 domain profile.
Similarity search - Domain/homology
RNA / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsFrazier, M.N. / Dillard, L.B. / Krahn, J.M. / Stanley, R.E.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIA ES103247 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES043010 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZI CES102488 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZI CES103206 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIC ES103326 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1ZIAES103340 United States
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Characterization of SARS2 Nsp15 nuclease activity reveals it's mad about U.
Authors: Meredith N Frazier / Lucas B Dillard / Juno M Krahn / Lalith Perera / Jason G Williams / Isha M Wilson / Zachary D Stewart / Monica C Pillon / Leesa J Deterding / Mario J Borgnia / Robin E Stanley /
Abstract: Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that ...Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3' of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3' of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.
History
DepositionMay 25, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 1, 2021Group: Database references / Category: citation / citation_author / database_2
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.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 6, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Uridylate-specific endoribonuclease
B: Uridylate-specific endoribonuclease
C: Uridylate-specific endoribonuclease
D: Uridylate-specific endoribonuclease
E: Uridylate-specific endoribonuclease
F: Uridylate-specific endoribonuclease
G: RNA (5'-R(*AP*UP*A)-3')
H: RNA (5'-R(*AP*UP*A)-3')
I: RNA (5'-R(*AP*UP*A)-3')
J: RNA (5'-R(*AP*UP*A)-3')
K: RNA (5'-R(*AP*UP*A)-3')
L: RNA (5'-R(*AP*UP*A)-3')


Theoretical massNumber of molelcules
Total (without water)260,66912
Polymers260,66912
Non-polymers00
Water9,998555
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "A"
d_3ens_1chain "C"
d_1ens_2chain "F"
d_2ens_2chain "E"
d_3ens_2chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLULEUC1 - 347
d_21ens_1GLULEUA1 - 347
d_31ens_1GLULEUE1 - 347
d_11ens_2GLULEUK1 - 347
d_21ens_2GLULEUI1 - 347
d_31ens_2GLULEUG1 - 347

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.50000030404, 0.866025227728, -2.99613772032E-5), (-0.866025208799, -0.500000285181, 0.000229223119951), (0.00018353230751, 0.000140558937617, 0.99999997328)30.2430129425, 140.130104557, -0.0243549119735
2given(-0.499936297478, -0.866062176791, 6.63082559252E-5), (0.866062179265, -0.499936296908, 2.60949873536E-5), (1.05500223709E-5, 7.04729039901E-5, 0.999999997461)136.479181848, 43.871110039, -0.00519272579724
3given(-0.500042439128, -0.866000874767, -0.000209699219931), (0.866000882681, -0.500042463023, 7.98080108377E-5), (-0.000173972321627, -0.000141692317157, 0.999999974828)136.506040836, 43.8761717111, 0.0186496367433
4given(-0.499893023585, 0.866086988384, 0.000541778050754), (-0.866087110974, -0.499892692946, -0.000641672521031), (-0.000284913332498, -0.000789994603456, 0.999999647366)30.1893625109, 140.183324446, 0.0762305023449

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Components

#1: Protein
Uridylate-specific endoribonuclease / NendoU / Non-structural protein 15 / nsp15


Mass: 42525.293 Da / Num. of mol.: 6
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 (E. coli)
References: UniProt: P0DTD1, Hydrolases; Acting on ester bonds
#2: RNA chain
RNA (5'-R(*AP*UP*A)-3')


Mass: 919.620 Da / Num. of mol.: 6 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 555 / Source method: isolated from a natural source / Formula: H2O

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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: Nsp15 viral endoribonuclease in a post-cleavage state / Type: COMPLEX / Details: Nsp15 with RNA 3-mer / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli (E. coli)
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 FIELD
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1058228 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 33.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007417025
ELECTRON MICROSCOPYf_angle_d0.617123130
ELECTRON MICROSCOPYf_chiral_restr0.05142664
ELECTRON MICROSCOPYf_plane_restr0.00422937
ELECTRON MICROSCOPYf_dihedral_angle_d14.43826156
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CELECTRON MICROSCOPYNCS constraints0.000705474832734
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.000708018618398
ens_2d_2KELECTRON MICROSCOPYNCS constraints0.000702633223939
ens_2d_3KELECTRON MICROSCOPYNCS constraints0.000698622032601

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