+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-24101 | |||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | SARS-CoV-2 Nsp15 endoribonuclease post-cleavage state | |||||||||||||||||||||
Map data | SARS-CoV-2 Nsp15 endoribonuclease post-cleavage state | |||||||||||||||||||||
Sample |
| |||||||||||||||||||||
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 / snRNP Assembly / TRAF3-dependent IRF activation pathway / Replication of the SARS-CoV-2 genome / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / : / double membrane vesicle viral factory outer membrane / 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 | |||||||||||||||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 / Homo sapiens (human) | |||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.2 Å | |||||||||||||||||||||
Authors | Frazier MN / Dillard LB / Krahn JM / Stanley RE | |||||||||||||||||||||
Funding support | United States, 6 items
| |||||||||||||||||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_24101.map.gz | 81.3 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-24101-v30.xml emd-24101.xml | 12.5 KB 12.5 KB | Display Display | EMDB header |
Images | emd_24101.png | 73.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-24101 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-24101 | HTTPS FTP |
-Related structure data
Related structure data | 7n06MC 7n33C M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_24101.map.gz / Format: CCP4 / Size: 99 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | SARS-CoV-2 Nsp15 endoribonuclease post-cleavage state | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.41168 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : Nsp15 viral endoribonuclease in a post-cleavage state
Entire | Name: Nsp15 viral endoribonuclease in a post-cleavage state |
---|---|
Components |
|
-Supramolecule #1: Nsp15 viral endoribonuclease in a post-cleavage state
Supramolecule | Name: Nsp15 viral endoribonuclease in a post-cleavage state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: Nsp15 with RNA 3-mer |
---|---|
Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 250 KDa |
-Macromolecule #1: Uridylate-specific endoribonuclease
Macromolecule | Name: 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 weight | Theoretical: 42.525293 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGSSHHHHHH SSGLVPRGSH MLEENLYFQS LEMSLENVAF NVVNKGHFDG QQGEVPVSII NNTVYTKVDG VDVELFENKT TLPVNVAFE LWAKRNIKPV PEVKILNNLG VDIAANTVIW DYKRDAPAHI STIGVCSMTD IAKKPTETIC APLTVFFDGR V DGQVDLFR ...String: MGSSHHHHHH SSGLVPRGSH MLEENLYFQS LEMSLENVAF NVVNKGHFDG QQGEVPVSII NNTVYTKVDG VDVELFENKT TLPVNVAFE LWAKRNIKPV PEVKILNNLG VDIAANTVIW DYKRDAPAHI STIGVCSMTD IAKKPTETIC APLTVFFDGR V DGQVDLFR NARNGVLITE GSVKGLQPSV GPKQASLNGV TLIGEAVKTQ FNYYKKVDGV VQQLPETYFT QSRNLQEFKP RS QMEIDFL ELAMDEFIER YKLEGYAFEH IVYGDFSHSQ LGGLHLLIGL AKRFKESPFE LEDFIPMDST VKNYFITDAQ TGS SKCVCS VIDLLLDDFV EIIKSQDLSV VSKVVKVTID YTEISFMLWC KDGHVETFYP KL |
-Macromolecule #2: RNA (5'-R(*AP*UP*A)-3')
Macromolecule | Name: RNA (5'-R(*AP*UP*A)-3') / type: rna / ID: 2 / Number of copies: 6 |
---|---|
Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 919.62 Da |
Sequence | String: AUA |
-Macromolecule #3: water
Macromolecule | Name: water / type: ligand / ID: 3 / Number of copies: 555 / Formula: HOH |
---|---|
Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
---|---|
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 54.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Initial angle assignment | Type: ANGULAR RECONSTITUTION |
---|---|
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1058228 |