+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-25915 | ||||||||||||
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Title | SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA | ||||||||||||
Map data | SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA | ||||||||||||
Sample |
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Keywords | endoribonuclease / 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 / Translation of Replicase and Assembly of the Replication Transcription Complex / TRAF3-dependent IRF activation pathway / 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 / symbiont-mediated suppression of host NF-kappaB cascade / 5'-3' DNA helicase activity / 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 / viral translational frameshifting / RNA-directed RNA polymerase / copper ion binding / 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 | ||||||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 / synthetic construct (others) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||||||||
Authors | Frazier MN / Krahn JM | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: bioRxiv / Year: 2022 Title: Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease. Authors: Meredith N Frazier / Isha M Wilson / Juno M Krahn / Kevin John Butay / Lucas B Dillard / Mario J Borgnia / Robin E Stanley / Abstract: Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, ...Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively. | ||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_25915.map.gz | 45 MB | EMDB map data format | |
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Header (meta data) | emd-25915-v30.xml emd-25915.xml | 12.8 KB 12.8 KB | Display Display | EMDB header |
Images | emd_25915.png | 89.4 KB | ||
Filedesc metadata | emd-25915.cif.gz | 5.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25915 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25915 | HTTPS FTP |
-Validation report
Summary document | emd_25915_validation.pdf.gz | 493 KB | Display | EMDB validaton report |
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Full document | emd_25915_full_validation.pdf.gz | 492.6 KB | Display | |
Data in XML | emd_25915_validation.xml.gz | 6.2 KB | Display | |
Data in CIF | emd_25915_validation.cif.gz | 7.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25915 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25915 | HTTPS FTP |
-Related structure data
Related structure data | 7tj2MC 7tqvC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_25915.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | SARS-CoV-2 endoribonuclease Nsp15 bound to dsRNA | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.91605 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : Structural Basis for dsRNA cleavage by the SARS-CoV-2 Endoribonuclease
Entire | Name: Structural Basis for dsRNA cleavage by the SARS-CoV-2 Endoribonuclease |
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Components |
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-Supramolecule #1: Structural Basis for dsRNA cleavage by the SARS-CoV-2 Endoribonuclease
Supramolecule | Name: Structural Basis for dsRNA cleavage by the SARS-CoV-2 Endoribonuclease type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Molecular weight | Theoretical: 250 KDa |
-Macromolecule #1: Uridylate-specific endoribonuclease nsp15
Macromolecule | Name: Uridylate-specific endoribonuclease nsp15 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: Lyases; Phosphorus-oxygen lyases |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Molecular weight | Theoretical: 39.246684 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #2: RNA (31-MER)
Macromolecule | Name: RNA (31-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 16.781023 KDa |
Sequence | String: GGAGGUAGUA GGUUGUAUAG UAGUAAGACC AGACCCUAGA CCAAUUCAUG CC |
-Macromolecule #3: RNA (31-MER)
Macromolecule | Name: RNA (31-MER) / type: rna / ID: 3 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 16.528768 KDa |
Sequence | String: GGCAUGAAUU GGUCUAGGGU CUGGUCUUAC UACUAUACAA CCUACUACCU CC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -1.2 µm / Nominal defocus min: 2.2 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 396312 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |