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- PDB-8p0u: Thogoto virus polymerase in Mode B conformation with defined endo... -

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Basic information

Entry
Database: PDB / ID: 8p0u
TitleThogoto virus polymerase in Mode B conformation with defined endonuclease domain and bound to 32-mer loop promoter RNA
Components
  • 3'RNA
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / Thogoto / viral RNA-dependent RNA polymerase
Function / homology
Function and homology information


cap snatching / 7-methylguanosine mRNA capping / virion component / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / host cell nucleus / RNA binding
Similarity search - Function
: / : / Polymerase basic protein 2 (PB2), '627' domain / Polymerase basic protein 2, cap-binding domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / Influenza RNA polymerase PB2 helical domain / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain ...: / : / Polymerase basic protein 2 (PB2), '627' domain / Polymerase basic protein 2, cap-binding domain / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / Influenza RNA polymerase PB2 helical domain / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein / Polymerase basic protein 2
Similarity search - Component
Biological speciesThogotovirus thogotoense
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsCusack, S. / Kouba, T.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research Agency France
CitationJournal: To Be Published
Title: Thogoto virus polymerase
Authors: Kouba, T. / Cusack, S.
History
DepositionMay 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 22, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
R: 3'RNA
V: 3'RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,6606
Polymers261,6355
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27540 Å2
ΔGint-198 kcal/mol
Surface area52790 Å2
MethodPISA

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Components

#1: Protein Polymerase acidic protein / PA / RNA-directed RNA polymerase subunit P3


Mass: 71551.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thogotovirus thogotoense / Gene: Segment 3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P27194
#2: Protein RNA-directed RNA polymerase catalytic subunit / RNA-directed RNA polymerase subunit P2


Mass: 81476.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thogotovirus thogotoense / Gene: Segment 2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O41353, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2 / PB2 / RNA-directed RNA polymerase subunit P3


Mass: 88172.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thogotovirus thogotoense / Gene: Segment 1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9YNA4
#4: RNA chain 3'RNA


Mass: 10217.090 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Thogotovirus thogotoense
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
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: Thogotovirus RNA-directed RNA polymerase / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Thogotovirus thogotoense
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 400 nm
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71517 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221897
ELECTRON MICROSCOPYf_angle_d0.4939594
ELECTRON MICROSCOPYf_dihedral_angle_d7.473697
ELECTRON MICROSCOPYf_chiral_restr0.0371710
ELECTRON MICROSCOPYf_plane_restr0.0023135

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