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- EMDB-24137: SARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state -

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Entry
Database: EMDB / ID: EMD-24137
TitleSARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state
Map dataSARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state
Sample
  • Complex: SARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state
    • Protein or peptide: Uridylate-specific endoribonuclease
  • RNA: RNA (5'-R(*A)-D(*(UFT))-R(P*A)-3')
Keywordsendoribonuclease / VIRAL PROTEIN / VIRAL PROTEIN-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 / snRNP Assembly / Replication of the SARS-CoV-2 genome / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / double membrane vesicle viral factory outer membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / SARS coronavirus main proteinase / 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 / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / SARS-CoV-2 modulates host translation machinery / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / mRNA (nucleoside-2'-O-)-methyltransferase activity / ubiquitinyl hydrolase 1 / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / viral protein processing / host cell endoplasmic reticulum membrane / lyase activity / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated activation 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 / : / 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 Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface 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 / (+) 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 / 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 / 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 replicase NSP7 / Peptidase family C16 domain profile. / 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 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsFrazier MN / Dillard LB
Funding support United States, 6 items
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 31, 2021-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
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  • Surface view with fitted model
  • Atomic models: PDB-7n33
  • Surface level: 0.3
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24137.png

Downloads & links

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Map

FileDownload / File: emd_24137.map.gz / Format: CCP4 / Size: 91.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.41 Å/pix.
x 314 pix.
= 128.411 Å		0.41 Å/pix.
x 278 pix.
= 113.689 Å		0.41 Å/pix.
x 276 pix.
= 112.871 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.40895 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.3
Minimum - Maximum-0.9120393 - 2.035604
Average (Standard dev.)0.038765863 (±0.160712)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin118120107
Dimensions278276314
Spacing276278314
CellA: 112.87089 Å / B: 113.6888 Å / C: 128.41109 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.408952898550720.408953237410070.40895222929936
M x/y/z276278314
origin x/y/z0.0000.0000.000
length x/y/z112.871113.689128.411
α/β/γ90.00090.00090.000
start NX/NY/NZ-288-288-288
NX/NY/NZ576576576
MAP C/R/S123
start NC/NR/NS120118107
NC/NR/NS276278314
D min/max/mean-0.9122.0360.039

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Supplemental data

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Sample components

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Entire : SARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state

EntireName: SARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state
Components
  • Complex: SARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state
    • Protein or peptide: Uridylate-specific endoribonuclease
  • RNA: RNA (5'-R(*A)-D(*(UFT))-R(P*A)-3')

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Supramolecule #1: SARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state

SupramoleculeName: SARS-CoV-2 Nsp15 endoribonuclease pre-cleavage state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: Uridylate-specific endoribonuclease

MacromoleculeName: Uridylate-specific endoribonuclease / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 40.523051 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMLEENLY FQSLEMSLEN VAFNVVNKGH FDGQQGEVPV SIINNTVYTK VDGVDVELFE NKTTLPVNVA FELWAKRNIK PVPEVKILN NLGVDIAANT VIWDYKRDAP AHISTIGVCS MTDIAKKPTE TICAPLTVFF DGRVDGQVDL FRNARNGVLI T EGSVKGLQ ...String:
GSHMLEENLY FQSLEMSLEN VAFNVVNKGH FDGQQGEVPV SIINNTVYTK VDGVDVELFE NKTTLPVNVA FELWAKRNIK PVPEVKILN NLGVDIAANT VIWDYKRDAP AHISTIGVCS MTDIAKKPTE TICAPLTVFF DGRVDGQVDL FRNARNGVLI T EGSVKGLQ PSVGPKQASL NGVTLIGEAV KTQFNYYKKV DGVVQQLPET YFTQSRNLQE FKPRSQMEID FLELAMDEFI ER YKLEGYA FEHIVYGDFS HSQLGGLHLL IGLAKRFKES PFELEDFIPM DSTVKNYFIT DAQTGSSKCV CSVIDLLLDD FVE IIKSQD LSVVSKVVKV TIDYTEISFM LWCKDGHVET FYPK

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #2: RNA (5'-R(*A)-D(*(UFT))-R(P*A)-3')

MacromoleculeName: RNA (5'-R(*A)-D(*(UFT))-R(P*A)-3') / type: rna / ID: 2 / Number of copies: 6
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 921.611 Da
SequenceString:
A(UFT)A

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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 K2 SUMMIT (4k x 4k) / Average electron dose: 54.0 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

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 383275
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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