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- EMDB-29779: Porcine epidemic diarrhea virus core polymerase complex -

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Entry
Database: EMDB / ID: EMD-29779
TitlePorcine epidemic diarrhea virus core polymerase complex
Map data
Sample
  • Complex: Core replication complex of porcine epidemic diarrhea virus composed of nsp12, nsp7, nsp8, and a short RNA substrate
    • Protein or peptide: nsp12
    • Protein or peptide: nsp8
    • Protein or peptide: nsp7
    • RNA: 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: 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')
  • Ligand: ZINC ION
KeywordsCoronavirus / nsp12 / RNA polymerase / PEDV / REPLICATION
Function / homology
Function and homology information


host cell membrane / endoplasmic reticulum-Golgi intermediate compartment / viral genome replication / 3'-5'-RNA exonuclease activity / host cell endoplasmic reticulum-Golgi intermediate compartment / 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 ...host cell membrane / endoplasmic reticulum-Golgi intermediate compartment / viral genome replication / 3'-5'-RNA exonuclease activity / host cell endoplasmic reticulum-Golgi intermediate compartment / 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 / identical protein binding
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
Replicase polyprotein 1ab / Replicase polyprotein 1ab / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesPorcine epidemic diarrhea virus / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAnderson TK / Kirchdoerfer RN
Funding support United States, 3 items
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, 2023-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29779.png

Downloads & links

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Map

FileDownload / File: emd_29779.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.2759882 - 0.47688708
Average (Standard dev.)0.0010125551 (±0.015787225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.504 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29779_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29779_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_29779_half_map_2.map
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Sample components

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Entire : Core replication complex of porcine epidemic diarrhea virus compo...

EntireName: Core replication complex of porcine epidemic diarrhea virus composed of nsp12, nsp7, nsp8, and a short RNA substrate
Components
  • Complex: Core replication complex of porcine epidemic diarrhea virus composed of nsp12, nsp7, nsp8, and a short RNA substrate
    • Protein or peptide: nsp12
    • Protein or peptide: nsp8
    • Protein or peptide: nsp7
    • RNA: 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: 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')
  • Ligand: ZINC ION

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Supramolecule #1: Core replication complex of porcine epidemic diarrhea virus compo...

SupramoleculeName: Core replication complex of porcine epidemic diarrhea virus composed of nsp12, nsp7, nsp8, and a short RNA substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Porcine epidemic diarrhea virus
Molecular weightTheoretical: 185 KDa

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Macromolecule #1: nsp12

MacromoleculeName: nsp12 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Porcine epidemic diarrhea virus
Molecular weightTheoretical: 104.939398 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DMAYLNRVRG SSAARLEPCN GTDTQHVYRA FDIYNKDVAC LGKFLKVNCV RLKNLDKHDA FYVVKRCTKS AMEHEQSIYS RLEKCGAVA EHDFFTWKDG RAIYGNVCRK DLTEYTMMDL CYALRNFDEN NCDVLKSILI KVGACEESYF NNKVWFDPVE N EDIHRVYA ...String:
DMAYLNRVRG SSAARLEPCN GTDTQHVYRA FDIYNKDVAC LGKFLKVNCV RLKNLDKHDA FYVVKRCTKS AMEHEQSIYS RLEKCGAVA EHDFFTWKDG RAIYGNVCRK DLTEYTMMDL CYALRNFDEN NCDVLKSILI KVGACEESYF NNKVWFDPVE N EDIHRVYA LLGTIVSRAM LKCVKFCDAM VEQGIVGVVT LDNQDLNGDF YDFGDFTCSI KGMGIPICTS YYSYMMPVMG MT NCLASEC FVKSDIFGED FKSYDLLEYD FTEHKTALFN KYFKYWGLQY HPNCVDCSDE QCIVHCANFN TLFSTTIPIT AFG PLCRKC WIDGVPLVTT AGYHFKQLGI VWNNDLNLHS SRLSINELLQ FCSDPALLIA SSPALVDQRT VCFSVAALGT GMTN QTVKP GHFNKEFYDF LLEQGFFSEG SELTLKHFFF AQKGDAAVKD FDYYRYNRPT VLDICQARVV YQIVQRYFDI YEGGC ITAK EVVVTNLNKS AGYPLNKFGK AGLYYESLSY EEQDELYAYT KRNILPTMTQ LNLKYAISGK ERARTVGGVS LLSTMT TRQ YHQKHLKSIV NTRGASVVIG TTKFYGGWDN MLKNLIDGVE NPCLMGWDYP KCDRALPNMI RMISAMILGS KHTTCCS ST DRFFRLCNEL AQVLTEVVYS NGGFYLKPGG TTSGDATTAY ANSVFNIFQA VSANVNKLLS VDSNVCHNLE VKQLQRKL Y ECCYRSTTVD DQFVVEYYGY LRKHFSMMIL SDDGVVCYNN DYASLGYVAD LNAFKAVLYY QNNVFMSASK CWIEPDINK GPHEFCSQHT MQIVDKDGTY YLPYPDPSRI LSAGVFVDDV VKTDAVVLLE RYVSLAIDAY PLSKHENPEY KKVFYVLLDW VKHLYKTLN AGVLESFSVT LLEDSTAKFW DESFYANMYE KS

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #2: nsp8

MacromoleculeName: nsp8 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Porcine epidemic diarrhea virus
Molecular weightTheoretical: 12.561573 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
RKSKVVSAMH SLLFGMLRRL DMSSVDTILN LAKDGVVPLS VIPAVSATKL NIVTSDIDSY NRIQREGCVH YAGTIWNIID IKDNDGKVV HVKEVTAQNA ESLSWPLVLG CERIV

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #3: nsp7

MacromoleculeName: nsp7 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Porcine epidemic diarrhea virus
Molecular weightTheoretical: 6.88201 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
KLTDIKCSNV VLLGCLSSMN VSANSTEWAY CVDLHNKINL CNDPEKAQEM LLALLAFFLS KN

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #4: RNA (5'-R(P*AP*AP*GP*AP*AP*GP*CP*UP*AP*UP*UP*AP*AP*AP*AP*UP*CP*AP...

MacromoleculeName: 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')
type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.406927 KDa
SequenceString:
AAGAAGCUAU UAAAAUCACA

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Macromolecule #5: RNA (5'-R(P*GP*GP*UP*UP*GP*UP*GP*AP*UP*UP*UP*UP*AP*AP*UP*AP*GP*CP...

MacromoleculeName: 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')
type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.364735 KDa
SequenceString:
GGUUGUGAUU UUAAUAGCUU

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNaClSodium chloridesodium chloride
25.0 mMHEPESHEPES
2.0 mMMgCl2Magnesium Chloride
2.0 mMDTTDithiothreitol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 1.2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 895007
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7cyq

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 74367
FSC plot (resolution estimation)

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