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- PDB-8g6r: Porcine epidemic diarrhea virus core polymerase complex -

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Basic information

Entry
Database: PDB / ID: 8g6r
TitlePorcine epidemic diarrhea virus core polymerase complex
Components
  • RNA (5'-R(P*AP*AP*GP*AP*AP*GP*CP*UP*AP*UP*UP*AP*AP*AP*AP*UP*CP*AP*CP*A)-3')
  • RNA (5'-R(P*GP*GP*UP*UP*GP*UP*GP*AP*UP*UP*UP*UP*AP*AP*UP*AP*GP*CP*UP*U)-3')
  • nsp12
  • nsp7
  • nsp8
KeywordsREPLICATION / Coronavirus / nsp12 / RNA polymerase / PEDV
Function / homology
Function and homology information


endoplasmic reticulum-Golgi intermediate compartment / host cell membrane / viral genome replication / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / symbiont-mediated perturbation of host ubiquitin-like protein modification / endonuclease activity / mRNA (nucleoside-2'-O-)-methyltransferase activity ...endoplasmic reticulum-Golgi intermediate compartment / host cell membrane / viral genome replication / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / symbiont-mediated perturbation of host ubiquitin-like protein modification / endonuclease activity / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / cysteine-type deubiquitinase activity / RNA helicase activity / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / induction by virus of host autophagy / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / proteolysis / RNA binding / zinc ion binding / ATP binding / identical protein binding / membrane
Similarity search - Function
Nonstructural protein 14, alphacoronavirus / RNA-dependent RNA polymerase, alphacoronavirus / Non-structural protein 5, alphacoronavirus / Non-structural protein 6, alphacoronavirus / Nonstructural protein 2, HCoV-229E-like / AAA domain / Pectin lyase fold/virulence factor / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus ...Nonstructural protein 14, alphacoronavirus / RNA-dependent RNA polymerase, alphacoronavirus / Non-structural protein 5, alphacoronavirus / Non-structural protein 6, alphacoronavirus / Nonstructural protein 2, HCoV-229E-like / AAA domain / Pectin lyase fold/virulence factor / 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 / 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 / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / NSP1, globular domain, alpha/betacoronavirus / 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 / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Papain-like protease, thumb domain superfamily, coronavirus / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Non-structural protein NSP7, coronavirus / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP8, coronavirus / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP7 superfamily, coronavirus / Non-structural protein NSP8 superfamily, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Peptidase C30, domain 3, coronavirus / Non-structural protein 6, coronavirus / Coronavirus replicase NSP3, C-terminal / Non-structural protein NSP4, N-terminal, coronavirus / Coronavirus endopeptidase C30 / Coronavirus papain-like peptidase / Coronavirus replicase NSP8 / Coronavirus RNA synthesis protein NSP10 / Coronavirus replicase NSP4, C-terminal / Coronavirus replicase NSP6 / Coronavirus replicase NSP4, N-terminal / Coronavirus replicase NSP3, C-terminal / Coronavirus main protease (M-pro) domain profile. / Coronavirus replicase NSP9 / Non-structural protein 3, X-domain-like / Macro domain / Appr-1"-p processing enzyme / Macro domain / Macro domain profile. / Macro domain-like / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Peptidase S1, PA clan, chymotrypsin-like fold
Similarity search - Domain/homology
RNA / RNA (> 10) / Replicase polyprotein 1ab / Replicase polyprotein 1ab / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesPorcine epidemic diarrhea virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAnderson, T.K. / Kirchdoerfer, R.N.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI123498 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI158463 United States
United States Department of Agriculture (USDA)WIS03099 United States
CitationJournal: bioRxiv / Year: 2023
Title: An alphacoronavirus polymerase structure reveals conserved co-factor functions.
Authors: Thomas K Anderson / Peter J Hoferle / Kenneth W Lee / Joshua J Coon / Robert N Kirchdoerfer /
Abstract: Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral ...Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from the betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine the structure of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. Our structure shows an unexpected nsp8 stoichiometry in comparison to other published coronavirus polymerase structures. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for RNA synthesis for alpha and betacoronaviruses as previously hypothesized. Our work shows the importance of studying diverse coronaviruses to reveal aspects of coronavirus replication while also identifying areas of conservation to be targeted by antiviral drugs.
History
DepositionFeb 15, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 4, 2023Group: Data collection / Data processing
Category: chem_comp_atom / chem_comp_bond / em_3d_reconstruction
Item: _em_3d_reconstruction.resolution

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: nsp12
B: nsp8
C: nsp7
I: RNA (5'-R(P*AP*AP*GP*AP*AP*GP*CP*UP*AP*UP*UP*AP*AP*AP*AP*UP*CP*AP*CP*A)-3')
J: RNA (5'-R(P*GP*GP*UP*UP*GP*UP*GP*AP*UP*UP*UP*UP*AP*AP*UP*AP*GP*CP*UP*U)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,2857
Polymers137,1555
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein nsp12


Mass: 104939.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine epidemic diarrhea virus / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0U2C263
#2: Protein nsp8


Mass: 12561.573 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine epidemic diarrhea virus / Gene: ORF1a, 1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1Z2R8Q6
#3: Protein nsp7


Mass: 6882.010 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine epidemic diarrhea virus / Gene: ORF1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0M4AW09

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RNA chain , 2 types, 2 molecules IJ

#4: RNA chain RNA (5'-R(P*AP*AP*GP*AP*AP*GP*CP*UP*AP*UP*UP*AP*AP*AP*AP*UP*CP*AP*CP*A)-3')


Mass: 6406.927 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: RNA chain RNA (5'-R(P*GP*GP*UP*UP*GP*UP*GP*AP*UP*UP*UP*UP*AP*AP*UP*AP*GP*CP*UP*U)-3')


Mass: 6364.735 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 2 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Core replication complex of porcine epidemic diarrhea virus composed of nsp12, nsp7, nsp8, and a short RNA substrate
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.185 MDa / Experimental value: NO
Source (natural)Organism: Porcine epidemic diarrhea virus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaCl1
225 mMHEPESHEPES1
32 mMMagnesium ChlorideMgCl21
42 mMDithiothreitolDTT1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
4cryoSPARC3.3.1CTF correction
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 895007
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74367 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059694
ELECTRON MICROSCOPYf_angle_d0.49113319
ELECTRON MICROSCOPYf_dihedral_angle_d7.1661645
ELECTRON MICROSCOPYf_chiral_restr0.0361516
ELECTRON MICROSCOPYf_plane_restr0.0031545

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