+Open data
-Basic information
Entry | Database: PDB / ID: 8q3b | ||||||||||||
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Title | The closed state of the ASFV apo-RNA polymerase | ||||||||||||
Components |
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Keywords | TRANSCRIPTION / RNA polymerase / ASFV / eukaryotic virus | ||||||||||||
Function / homology | Function and homology information DNA-templated viral transcription / RNA polymerase III activity / viral transcription / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity ...DNA-templated viral transcription / RNA polymerase III activity / viral transcription / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / host cell cytoplasm / protein dimerization activity / DNA-templated transcription / DNA binding / zinc ion binding / metal ion binding Similarity search - Function | ||||||||||||
Biological species | African swine fever virus BA71V | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.69 Å | ||||||||||||
Authors | Pilotto, S. / Sykora, M. / Cackett, G. / Werner, F. | ||||||||||||
Funding support | United Kingdom, 3items
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Citation | Journal: Nat Commun / Year: 2024 Title: Structure of the recombinant RNA polymerase from African Swine Fever Virus. Authors: Simona Pilotto / Michal Sýkora / Gwenny Cackett / Christopher Dulson / Finn Werner / Abstract: African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of ...African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8q3b.cif.gz | 743.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8q3b.ent.gz | 592.6 KB | Display | PDB format |
PDBx/mmJSON format | 8q3b.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8q3b_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 8q3b_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 8q3b_validation.xml.gz | 101.2 KB | Display | |
Data in CIF | 8q3b_validation.cif.gz | 155.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/q3/8q3b ftp://data.pdbj.org/pub/pdb/validation_reports/q3/8q3b | HTTPS FTP |
-Related structure data
Related structure data | 18120MC 8q3kC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA-directed RNA polymerase ... , 7 types, 7 molecules ABCDEFJ
#1: Protein | Mass: 163936.094 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-99, NP1450L / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P42486, DNA-directed RNA polymerase |
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#2: Protein | Mass: 140119.406 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: The protein was tagged at the N-terminus with a His-ZZ cleavable tag. The tag was cleaved with TEV protease leaving a G as the first residue Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-053 / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P42487 |
#3: Protein | Mass: 41352.035 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-113 / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q65184 |
#4: Protein | Mass: 38789.609 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-105 / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q89907 |
#5: Protein | Mass: 23693.711 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-108 / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q65181 |
#6: Protein | Mass: 16704.811 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-069 / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P42484 |
#8: Protein | Mass: 9098.826 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-95 / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P42488 |
-Protein , 1 types, 1 molecules I
#7: Protein | Mass: 11831.861 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) African swine fever virus BA71V / Gene: Ba71V-065 / Plasmid: pBIG2 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q65157 |
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-Non-polymers , 2 types, 7 molecules
#9: Chemical | ChemComp-ZN / #10: Chemical | ChemComp-MG / | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.45 MDa / Experimental value: YES | ||||||||||||||||||||||||||||||
Source (natural) | Organism: African swine fever virus BA71V | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Trichoplusia ni (cabbage looper) / Strain: High Five / Plasmid: pBIG2 | ||||||||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisparse | ||||||||||||||||||||||||||||||
Specimen support | Details: The grid was covered with freshly prepared graphene oxide prior to use Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.6 sec. / Electron dose: 48.152 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 14638 Details: Images were collected in movie-mode for a total of 50 frames per image. The data collection was carried out in super-resolution mode and binned 2 on-the-fly. |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
EM software |
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Image processing | Details: Images were motion corrected in Relion v4 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4842857 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 467000 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 113 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
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