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- PDB-8q3b: The closed state of the ASFV apo-RNA polymerase -

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Basic information

Entry
Database: PDB / ID: 8q3b
TitleThe closed state of the ASFV apo-RNA polymerase
Components
  • (DNA-directed RNA polymerase ...) x 7
  • Uncharacterized protein C122R
KeywordsTRANSCRIPTION / RNA polymerase / ASFV / eukaryotic virus
Function / homology
Function and homology information


DNA-templated viral transcription / viral transcription / RNA polymerase III activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / RNA polymerase II activity / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity ...DNA-templated viral transcription / viral transcription / RNA polymerase III activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / RNA polymerase II activity / 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
RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. ...RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
DNA-directed RNA polymerase RPB6 homolog / DNA-directed RNA polymerase RPB1 homolog / DNA-directed RNA polymerase RPB2 homolog / DNA-directed RNA polymerase RPB10 homolog / Uncharacterized protein C122R / DNA-directed RNA polymerase RPB5 homolog / DNA-directed RNA polymerase RPB3-11 homolog / DNA-directed RNA polymerase RPB7 homolog
Similarity search - Component
Biological speciesAfrican swine fever virus BA71V
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsPilotto, S. / Sykora, M. / Cackett, G. / Werner, F.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X017028/1 United Kingdom
Wellcome TrustWT207446/Z/17/Z United Kingdom
Wellcome TrustWT108877/B/15/Z United Kingdom
CitationJournal: 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
DepositionAug 3, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 14, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase RPB1 homolog
B: DNA-directed RNA polymerase RPB2 homolog
C: DNA-directed RNA polymerase RPB3-11 homolog
D: DNA-directed RNA polymerase RPB7 homolog
E: DNA-directed RNA polymerase RPB5 homolog
F: DNA-directed RNA polymerase RPB6 homolog
I: Uncharacterized protein C122R
J: DNA-directed RNA polymerase RPB10 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)445,94315
Polymers445,5268
Non-polymers4177
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, single particle cryo-EM
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA-directed RNA polymerase ... , 7 types, 7 molecules ABCDEFJ

#1: Protein DNA-directed RNA polymerase RPB1 homolog / RPB1 homolog


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
#2: Protein DNA-directed RNA polymerase RPB2 homolog


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 DNA-directed RNA polymerase RPB3-11 homolog


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 DNA-directed RNA polymerase RPB7 homolog


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 DNA-directed RNA polymerase RPB5 homolog


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 DNA-directed RNA polymerase RPB6 homolog


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 DNA-directed RNA polymerase RPB10 homolog


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

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Protein , 1 types, 1 molecules I

#7: Protein Uncharacterized protein C122R


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 / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / 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: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 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 solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtrisC4H11NO31
2250 mMsodium chlorideNaCl1
31 mMmagnesium chlorideMgCl21
40.1 mMzinc sulfateZnSO41
51 mMDTTC4H10O2S21
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisparse
Specimen supportDetails: 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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: 2700 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1Topazparticle selection
2EPU2image acquisition
4CTFFIND4CTF correction
7PHENIX1.2model fittingDock in map
9cryoSPARC3initial Euler assignment2/3D classifications and 3DVA
10RELION4initial Euler assignment3D refinement and post-processing
11cryoSPARC4final Euler assignment3D flexibility refinement
13cryoSPARC43D reconstruction3D flexibility refinement
14PHENIX1.2model refinementreal space refinement
15Coot0.9.8.7model refinementfor manual editing
Image processingDetails: Images were motion corrected in Relion v4
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4842857
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 467000 / Symmetry type: POINT
Atomic model buildingB value: 113 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00330012
ELECTRON MICROSCOPYf_angle_d0.54540660
ELECTRON MICROSCOPYf_dihedral_angle_d5.814059
ELECTRON MICROSCOPYf_chiral_restr0.0424638
ELECTRON MICROSCOPYf_plane_restr0.0045228

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