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- PDB-8psx: Tilapia Lake Virus polymerase in vRNA elongation state (transcrip... -

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Basic information

Entry
Database: PDB / ID: 8psx
TitleTilapia Lake Virus polymerase in vRNA elongation state (transcriptase conformation)
Components
  • 5' vRNA end - vRNA loop (40-mer)
  • Polymerase acidic protein (PA-like)
  • Putative PB1
  • RNA-dependent RNA polymerase
  • Transcription-like product
KeywordsVIRAL PROTEIN / Viral polymerase
Function / homologyRNA-dependent RNA polymerase activity / DNA/RNA polymerase superfamily / Chem-A0I / RNA / RNA (> 10) / Uncharacterized protein / Putative PB1 / RNA-dependent RNA polymerase
Function and homology information
Biological speciesTilapia lake virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsArragain, B. / Cusack, S.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-18-CE11-0028 France
CitationJournal: Nat Commun / Year: 2023
Title: Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family.
Authors: Benoit Arragain / Martin Pelosse / Albert Thompson / Stephen Cusack /
Abstract: Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ...Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ends and encode proteins with no known homologues, apart from the segment 1, which encodes an orthomyxo-like RNA-dependent-RNA polymerase core subunit. Here we show that segments 1-3 encode respectively the PB1, PB2 and PA-like subunits of an active heterotrimeric polymerase that maintains all domains found in the distantly related influenza polymerase, despite an unprecedented overall size reduction of 40%. Multiple high-resolution cryo-EM structures of TiLV polymerase in pre-initiation, initiation and active elongation states, show how it binds the vRNA and cRNA promoters and performs RNA synthesis, with both transcriptase and replicase configurations being characterised. However, the highly truncated endonuclease-like domain appears inactive and the putative cap-binding domain is autoinhibited, emphasising that many functional aspects of TiLV polymerase remain to be elucidated.
History
DepositionJul 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 27, 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: Polymerase acidic protein (PA-like)
B: Putative PB1
C: RNA-dependent RNA polymerase
V: 5' vRNA end - vRNA loop (40-mer)
S: 5' vRNA end - vRNA loop (40-mer)
P: Transcription-like product
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,61712
Polymers190,8906
Non-polymers7276
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29410 Å2
ΔGint-175 kcal/mol
Surface area59180 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Polymerase acidic protein (PA-like)


Mass: 47780.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tilapia lake virus / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A142I7Z3
#2: Protein Putative PB1


Mass: 57179.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tilapia lake virus / Gene: PB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A1Y9SHW4
#3: Protein RNA-dependent RNA polymerase /


Mass: 53782.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tilapia lake virus / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A7G3S745

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RNA chain , 2 types, 3 molecules VSP

#4: RNA chain 5' vRNA end - vRNA loop (40-mer)


Mass: 12736.518 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Tilapia lake virus
#5: RNA chain Transcription-like product


Mass: 6675.037 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tilapia lake virus

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Non-polymers , 3 types, 6 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-A0I / [(2~{R},3~{S},4~{R},5~{R})-5-(4-azanyl-2-oxidanylidene-pyrimidin-1-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-~{N}-[oxidanyl(phosphonooxy)phosphoryl]phosphonamidic acid


Mass: 482.172 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H17N4O13P3 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / 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: Tilapia lake virus polymerase / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Tilapia lake virus
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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29892 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311797
ELECTRON MICROSCOPYf_angle_d0.48316128
ELECTRON MICROSCOPYf_dihedral_angle_d6.4481951
ELECTRON MICROSCOPYf_chiral_restr0.0361830
ELECTRON MICROSCOPYf_plane_restr0.0041938

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