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- PDB-8yax: SARS-CoV-2 DMV nsp3-4 pore complex (full-pore) -

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Basic information

Entry
Database: PDB / ID: 8yax
TitleSARS-CoV-2 DMV nsp3-4 pore complex (full-pore)
Components
  • Non-structural protein 4
  • Papain-like protease nsp3
KeywordsVIRAL PROTEIN / Double membrane vesicle / pore complex / nsp3 / nsp4 / RNA transport
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 / 5'-3' DNA helicase activity / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / SARS coronavirus main proteinase / host cell endosome / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / symbiont-mediated degradation of host mRNA / symbiont-mediated suppression of host toll-like receptor signaling pathway / 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 / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / mRNA (nucleoside-2'-O-)-methyltransferase activity / DNA helicase / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / viral protein processing / lyase activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / RNA helicase / induction by virus of host autophagy / copper ion binding / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / 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 / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / : / Coronavirus Nsp12 Interface domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / : / Coronavirus Nsp12 Interface domain profile. / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / NSP12 RNA-dependent RNA polymerase, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus 2-O-methyltransferase / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nonstructural protein 13, 1B domain, coronavirus / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / 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 / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) 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 / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / DNA2/NAM7 helicase-like, C-terminal / AAA domain / Lipocalin signature. / (+) 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 Nsp3e group 2-specific marker (G2M) domain profile. / NSP1, C-terminal domain, betacoronavirus / 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 / Betacoronavirus (BetaCoV) Nsp1 C-terminal 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 superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / : / Papain-like protease, N-terminal domain superfamily, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / Coronavirus 3Ecto domain profile. / : / Coronavirus (CoV) Nsp2 middle domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / NSP1, globular domain, alpha/betacoronavirus / : / Coronavirus (CoV) Nsp3 Y domain profile. / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus replicase NSP2, N-terminal / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus
Similarity search - Domain/homology
Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 4.9 Å
AuthorsHuang, Y.X. / Zhong, L.J. / Zhang, W.X. / Ni, T.
Funding support Hong Kong, 1items
OrganizationGrant numberCountry
The University Grants Committee, Research Grants Council (RGC) Hong Kong
CitationJournal: To Be Published
Title: Molecular Architecture of Coronavirus Double Membrane Vesicle Pore Complex
Authors: Huang, Y.X. / Ni, T.
History
DepositionFeb 10, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Papain-like protease nsp3
B: Papain-like protease nsp3
C: Non-structural protein 4
D: Non-structural protein 4


Theoretical massNumber of molelcules
Total (without water)547,4024
Polymers547,4024
Non-polymers00
Water00
1
A: Papain-like protease nsp3
B: Papain-like protease nsp3
C: Non-structural protein 4
D: Non-structural protein 4
x 6


Theoretical massNumber of molelcules
Total (without water)3,284,40924
Polymers3,284,40924
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5

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Components

#1: Protein Papain-like protease nsp3 / Non-structural protein 3 / nsp3 / PL2-PRO / Papain-like proteinase / PL-PRO


Mass: 217471.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: P0DTD1, ubiquitinyl hydrolase 1
#2: Protein Non-structural protein 4 / nsp4


Mass: 56229.582 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: P0DTD1

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: SARS-CoV-2 nsp3-4 pore complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293F
Buffer solutionpH: 8 / Details: 150mM NaCl, 10mM Tris-HCl, 1mM EDTA
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
210 mMTris hydrochlorideTris-HCl1
31 mMEthylenediaminetetraacetic acidEDTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 6000 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 3 e/Å2 / Avg electron dose per subtomogram: 105 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
Details: The tilt-series were acquired using Thermofisher Krios equipped with a Falcon 4i camera and Selectris energy filter. A dose-symmetric scheme (group of 2) was used, with a tilt range of -51 ...Details: The tilt-series were acquired using Thermofisher Krios equipped with a Falcon 4i camera and Selectris energy filter. A dose-symmetric scheme (group of 2) was used, with a tilt range of -51 degree to 51 degree (or -60 to 60) at 3 degree increments and an exposure dose of 3 e/A2 per image. The total dose was 105 or 123 e/A2.

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Processing

EM software
IDNameVersionCategory
1emClarityv1.6.2volume selection
4emClarityv1.6.2CTF correction
5RELIONv4CTF correction
8UCSF Chimera1.16model fitting
9UCSF ChimeraX1.6.1model fitting
10Coot0.9.8model fitting
11ISOLDE1.6.0model fitting
14RELIONv4final Euler assignment
15RELIONv4classification
16RELIONv43D reconstruction
17PHENIX1.21_5207model refinement
Image processingDetails: Falcon 4i Selectris X energy filter
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30607 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 4746 / Num. of volumes extracted: 598699 / Reference model: EMD-11514
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
18F2E

8f2e

18F2E2PDBexperimental model
26WRH

6wrh

16WRH3PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 381.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001420956
ELECTRON MICROSCOPYf_angle_d0.439228502
ELECTRON MICROSCOPYf_chiral_restr0.03783279
ELECTRON MICROSCOPYf_plane_restr0.00353571
ELECTRON MICROSCOPYf_dihedral_angle_d9.38267435

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