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- EMDB-44590: SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotid... -

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Entry
Database: EMDB / ID: EMD-44590
TitleSARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotide bulge
Map dataSARS-CoV-2 Nsp15 with bound dsRNA, which contains a 1 nucleotide bulge unpaired U
Sample
  • Complex: SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotide bulge
    • Protein or peptide: Uridylate-specific endoribonuclease nsp15
    • RNA: RNA (35-mer)
    • RNA: RNA (34-mer)
KeywordsUridine-specific endonuclease / bulged dsRNA / unpaired nucleotide / coronavirus / VIRAL PROTEIN
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: 3.24 Å
AuthorsWright ZM / Butay KJ / Krahn JM / Borgnia MJ / Stanley RE
Funding support United States, 3 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)ZIC ES103326 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1ZIAES103340 United States
CitationJournal: Nat Commun / Year: 2025
Title: Spontaneous base flipping helps drive Nsp15's preferences in double stranded RNA substrates.
Authors: Zoe M Wright / Kevin John Butay / Juno M Krahn / Isha M Wilson / Scott A Gabel / Eugene F DeRose / Israa S Hissein / Jason G Williams / Mario J Borgnia / Meredith N Frazier / Geoffrey A ...Authors: Zoe M Wright / Kevin John Butay / Juno M Krahn / Isha M Wilson / Scott A Gabel / Eugene F DeRose / Israa S Hissein / Jason G Williams / Mario J Borgnia / Meredith N Frazier / Geoffrey A Mueller / Robin E Stanley /
Abstract: Coronaviruses evade detection by the host immune system with the help of the endoribonuclease Nsp15, which regulates levels of viral double stranded RNA by cleaving 3' of uridine (U). While prior ...Coronaviruses evade detection by the host immune system with the help of the endoribonuclease Nsp15, which regulates levels of viral double stranded RNA by cleaving 3' of uridine (U). While prior structural data shows that to cleave double stranded RNA, Nsp15's target U must be flipped out of the helix, it is not yet understood whether Nsp15 initiates flipping or captures spontaneously flipped bases. We address this gap by designing fluorinated double stranded RNA substrates that allow us to directly relate a U's sequence context to both its tendency to spontaneously flip and its susceptibility to cleavage by Nsp15. Through a combination of nuclease assays, F NMR spectroscopy, mass spectrometry, and single particle cryo-EM, we determine that Nsp15 acts most efficiently on unpaired Us, particularly those that are already flipped. Across sequence contexts, we find Nsp15's cleavage efficiency to be directly related to that U's tendency to spontaneously flip. Overall, our findings unify previous characterizations of Nsp15's cleavage preferences, and suggest that activity of Nsp15 during infection is partially driven by bulged or otherwise relatively accessible Us that appear at strategic positions in the viral RNA.
History
DepositionApr 23, 2024-
Header (metadata) releaseDec 18, 2024-
Map releaseDec 18, 2024-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44590.png

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Map

FileDownload / File: emd_44590.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSARS-CoV-2 Nsp15 with bound dsRNA, which contains a 1 nucleotide bulge unpaired U
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.29 Å/pix.
x 210 pix.
= 271.803 Å		1.29 Å/pix.
x 210 pix.
= 271.803 Å		1.29 Å/pix.
x 210 pix.
= 271.803 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2943 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.257
Minimum - Maximum-0.5995911 - 1.8915013
Average (Standard dev.)-0.0021254066 (±0.06572269)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 271.80298 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44590_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_44590_half_map_2.map
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Sample components

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Entire : SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotid...

EntireName: SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotide bulge
Components
  • Complex: SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotide bulge
    • Protein or peptide: Uridylate-specific endoribonuclease nsp15
    • RNA: RNA (35-mer)
    • RNA: RNA (34-mer)

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Supramolecule #1: SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotid...

SupramoleculeName: SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA with 1 nucleotide bulge
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Macromolecule #1: Uridylate-specific endoribonuclease nsp15

MacromoleculeName: Uridylate-specific endoribonuclease nsp15 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: Lyases; Phosphorus-oxygen lyases
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 39.246684 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SLEMSLENVA FNVVNKGHFD GQQGEVPVSI INNTVYTKVD GVDVELFENK TTLPVNVAFE LWAKRNIKPV PEVKILNNLG VDIAANTVI WDYKRDAPAH ISTIGVCSMT DIAKKPTETI CAPLTVFFDG RVDGQVDLFR NARNGVLITE GSVKGLQPSV G PKQASLNG ...String:
SLEMSLENVA FNVVNKGHFD GQQGEVPVSI INNTVYTKVD GVDVELFENK TTLPVNVAFE LWAKRNIKPV PEVKILNNLG VDIAANTVI WDYKRDAPAH ISTIGVCSMT DIAKKPTETI CAPLTVFFDG RVDGQVDLFR NARNGVLITE GSVKGLQPSV G PKQASLNG VTLIGEAVKT QFNYYKKVDG VVQQLPETYF TQSRNLQEFK PRSQMEIDFL ELAMDEFIER YKLEGYAFEA IV YGDFSHS QLGGLHLLIG LAKRFKESPF ELEDFIPMDS TVKNYFITDA QTGSSKCVCS VIDLLLDDFV EIIKSQDLSV VSK VVKVTI DYTEISFMLW CKDGHVETFY PKLQ

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #2: RNA (35-mer)

MacromoleculeName: RNA (35-mer) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 11.12766 KDa
SequenceString:
UUUAGAUUUC AUCUAAACGA ACAAACUAAA AUGUC

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Macromolecule #3: RNA (34-mer)

MacromoleculeName: RNA (34-mer) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 10.826366 KDa
SequenceString:
GACAUUUUAG UUUGUUCGUU UGAUGAAAUC UAAA

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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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
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: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 217900
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING

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