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- PDB-8urb: Porcine epidemic diarrhea virus complete core polymerase complex -

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Basic information

Entry
Database: PDB / ID: 8urb
TitlePorcine epidemic diarrhea virus complete core polymerase complex
Components
  • RNA (33-MER)
  • RNA (55-MER)
  • nsp12
  • nsp7
  • nsp8
KeywordsREPLICATION / TRANSFERASE/RNA / Coronavirus / nsp12 / RNA polymerase / PEDV / TRANSFERASE-RNA complex
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 / 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 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 / : / 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 / (+) RNA virus helicase core domain / (+)RNA virus helicase core 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 / 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
Similarity search - Domain/homology
RNA / RNA (> 10) / ORF1ab polyprotein
Similarity search - Component
Biological speciesPorcine epidemic diarrhea virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 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
DepositionOct 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release

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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
D: nsp8
I: RNA (33-MER)
J: RNA (55-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)189,9998
Polymers189,8686
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 4 molecules ABDC

#1: Protein nsp12


Mass: 108994.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine epidemic diarrhea virus / Gene: 1b, ORF1a / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: U6BRU0
#2: Protein nsp8


Mass: 21716.838 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine epidemic diarrhea virus / Gene: 1b, ORF1a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: U6BRU0
#3: Protein nsp7


Mass: 9230.511 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine epidemic diarrhea virus / Gene: 1b, ORF1a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: U6BRU0

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RNA chain , 2 types, 2 molecules IJ

#4: RNA chain RNA (33-MER)


Mass: 10468.266 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: RNA chain RNA (55-MER)


Mass: 17740.572 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 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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 polymerase complex of the 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: YES
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 mMHEPES1
32 mMMagnesium ChlorideMgCl21
42 mMdithiothreitol1
56 mMCHAPSO1
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategoryFitting-ID
1cryoSPARC4.1.0particle selection
4cryoSPARC4.1.0CTF correction
9cryoSPARC4.1.0initial Euler assignment
10cryoSPARC4.1.0final Euler assignment
11cryoSPARC4.1.0classification
12cryoSPARC4.1.03D reconstruction
13PHENIX1.20.1_4487:model refinement1
22ISOLDEmodel refinement2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103940 / Symmetry type: POINT
Atomic model building
ID
1
2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311960
ELECTRON MICROSCOPYf_angle_d0.48716390
ELECTRON MICROSCOPYf_dihedral_angle_d6.4061969
ELECTRON MICROSCOPYf_chiral_restr0.0361865
ELECTRON MICROSCOPYf_plane_restr0.0031947

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