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- EMDB-23786: Cryo-EM structure of SARS-CoV-2 NSP15 NendoU at pH 6.0 -

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Entry
Database: EMDB / ID: EMD-23786
TitleCryo-EM structure of SARS-CoV-2 NSP15 NendoU at pH 6.0
Map data
Sample
  • Complex: Structure of SARS-CoV-2 NSP15
    • Protein or peptide: Uridylate-specific endoribonuclease
Keywordsnsp15 / sars-cov-2 / nendoU / coronavirus / covid-19 / VIRAL PROTEIN / HYDROLASE
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) / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Maturation of replicase proteins / 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) / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Maturation of replicase proteins / ISG15-specific peptidase activity / TRAF3-dependent IRF activation pathway / Transcription of SARS-CoV-2 sgRNAs / snRNP Assembly / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-2 genome / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / host cell endoplasmic reticulum-Golgi intermediate compartment / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / 5'-3' DNA helicase activity / 3'-5'-RNA exonuclease activity / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / symbiont-mediated suppression of host toll-like receptor signaling pathway / G-quadruplex RNA binding / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / omega peptidase activity / mRNA (guanine-N7)-methyltransferase / SARS-CoV-2 modulates host translation machinery / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated suppression of host NF-kappaB cascade / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / regulation of autophagy / 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 / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell nucleus / 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: 2.48 Å
AuthorsGodoy AS / Song Y
Funding support Brazil, 1 items
OrganizationGrant numberCountry
Sao Paulo Research Foundation (FAPESP)2013/07600-3 Brazil
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease.
Authors: Andre Schutzer Godoy / Aline Minalli Nakamura / Alice Douangamath / Yun Song / Gabriela Dias Noske / Victor Oliveira Gawriljuk / Rafaela Sachetto Fernandes / Humberto D Muniz Pereira / ...Authors: Andre Schutzer Godoy / Aline Minalli Nakamura / Alice Douangamath / Yun Song / Gabriela Dias Noske / Victor Oliveira Gawriljuk / Rafaela Sachetto Fernandes / Humberto D Muniz Pereira / Ketllyn Irene Zagato Oliveira / Daren Fearon / Alexandre Dias / Tobias Krojer / Michael Fairhead / Alisa Powell / Louise Dunnet / Jose Brandao-Neto / Rachael Skyner / Rod Chalk / Dávid Bajusz / Miklós Bege / Anikó Borbás / György Miklós Keserű / Frank von Delft / Glaucius Oliva /
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and ...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
History
DepositionApr 6, 2021-
Header (metadata) releaseApr 14, 2021-
Map releaseApr 14, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

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  • Surface view with section colored by density value
  • Surface level: 4.55
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  • Surface view colored by cylindrical radius
  • Surface level: 4.55
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  • Surface view with fitted model
  • Atomic models: PDB-7me0
  • Surface level: 4.55
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

emd_23786.png

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Map

FileDownload / File: emd_23786.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 256 pix.
= 212.736 Å		0.83 Å/pix.
x 256 pix.
= 212.736 Å		0.83 Å/pix.
x 256 pix.
= 212.736 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.831 Å
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Contour LevelBy AUTHOR: 4.55 / Movie #1: 4.55
Minimum - Maximum-10.027505 - 22.832151
Average (Standard dev.)0.000000000001574 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.736 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8310.8310.831
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z212.736212.736212.736
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-10.02822.8320.000

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Supplemental data

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Mask #1

Fileemd_23786_msk_1.map
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Fileemd_23786_additional_1.map
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Half map: #1

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Sample components

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Entire : Structure of SARS-CoV-2 NSP15

EntireName: Structure of SARS-CoV-2 NSP15
Components
  • Complex: Structure of SARS-CoV-2 NSP15
    • Protein or peptide: Uridylate-specific endoribonuclease

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Supramolecule #1: Structure of SARS-CoV-2 NSP15

SupramoleculeName: Structure of SARS-CoV-2 NSP15 / 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

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: 42.117844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKHHHHHHPM SDYDIPTTEN LYFQGAMSLE NVAFNVVNKG HFDGQQGEVP VSIINNTVYT KVDGVDVELF ENKTTLPVNV AFELWAKRN IKPVPEVKIL NNLGVDIAAN TVIWDYKRDA PAHISTIGVC SMTDIAKKPT ETICAPLTVF FDGRVDGQVD L FRNARNGV ...String:
MKHHHHHHPM SDYDIPTTEN LYFQGAMSLE NVAFNVVNKG HFDGQQGEVP VSIINNTVYT KVDGVDVELF ENKTTLPVNV AFELWAKRN IKPVPEVKIL NNLGVDIAAN TVIWDYKRDA PAHISTIGVC SMTDIAKKPT ETICAPLTVF FDGRVDGQVD L FRNARNGV LITEGSVKGL QPSVGPKQAS LNGVTLIGEA VKTQFNYYKK VDGVVQQLPE TYFTQSRNLQ EFKPRSQMEI DF LELAMDE FIERYKLEGY AFEHIVYGDF SHSQLGGLHL LIGLAKRFKE SPFELEDFIP MDSTVKNYFI TDAQTGSSKC VCS VIDLLL DDFVEIIKSQ DLSVVSKVVK VTIDYTEISF MLWCKDGHVE TFYPKLQ

UniProtKB: Replicase polyprotein 1ab

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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

Concentration0.5 mg/mL
BufferpH: 6
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 41.70522228 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: PDB ENTRY
PDB model - PDB ID:

7keg

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 712742
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 100 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7keg


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7me0:
Cryo-EM structure of SARS-CoV-2 NSP15 NendoU at pH 6.0

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