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- PDB-9hby: TiLV-NP hexamer (pseudo-C6) (local refinement around 3 TiLV-NPs) -

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Basic information

Entry
Database: PDB / ID: 9hby
TitleTiLV-NP hexamer (pseudo-C6) (local refinement around 3 TiLV-NPs)
Components
  • 40-mer vRNA loop
  • Tilapia Lake Virus nucleoprotein (segment 4)
KeywordsRNA BINDING PROTEIN / Viral protein / nucleoprotein / oligomer
Function / homologyDNA / DNA (> 10) / Uncharacterized protein
Function and homology information
Biological speciesTilapia lake virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsArragain, B. / Cusack, S.
Funding support France, 1items
OrganizationGrant numberCountry
Other government France
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structure of the tilapia lake virus nucleoprotein bound to RNA.
Authors: Benoît Arragain / Martin Pelosse / Karine Huard / Stephen Cusack /
Abstract: Tilapia Lake virus (TiLV) belongs to the Amnoonviridae family within the Articulavirales order of segmented negative-strand RNA viruses and is highly diverged from more familiar orthomyxoviruses, ...Tilapia Lake virus (TiLV) belongs to the Amnoonviridae family within the Articulavirales order of segmented negative-strand RNA viruses and is highly diverged from more familiar orthomyxoviruses, such as influenza. The viral nucleoprotein (NP), a key component of the replication machinery, packages the viral genome into protective ribonucleoprotein particles. Here we describe the electron cryo-microscopy (cryo-EM) structure of TiLV-NP bound to RNA within in vitro reconstituted, small ring-like, pseudo-symmetrical oligomers. Although TiLV-NP is considerably smaller than its influenza counterpart and unrelated in sequence, it maintains the same topology and domain organisation. This comprises a head and body domain between which is a positively charged groove, where single-stranded RNA binds. In addition, an oligomerisation loop inserts into a hydrophobic pocket in the neighbouring NP, the flexible hinges of which allow variable orientation of adjacent NPs. Focused cryo-EM maps unambiguously define the 5' to 3' direction of the bound RNA, confirmed by double stranded, A-form RNA regions that extrude out from some of the NP-NP interfaces. This is the first fully resolved description of how single-stranded and stem-loop RNA binds to an articulaviral NP assembly. Superposition with orthomyxoviral NPs suggest that the mode of RNA binding is likely similar across the Articulavirales order.
History
DepositionNov 8, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tilapia Lake Virus nucleoprotein (segment 4)
C: Tilapia Lake Virus nucleoprotein (segment 4)
D: Tilapia Lake Virus nucleoprotein (segment 4)
E: Tilapia Lake Virus nucleoprotein (segment 4)
M: 40-mer vRNA loop
N: 40-mer vRNA loop
O: 40-mer vRNA loop


Theoretical massNumber of molelcules
Total (without water)200,6937
Polymers200,6937
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Tilapia Lake Virus nucleoprotein (segment 4)


Mass: 38387.129 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tilapia lake virus / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A1Y9SHW7
#2: DNA chain 40-mer vRNA loop


Mass: 15714.807 Da / Num. of mol.: 3 / Source method: obtained synthetically
Details: The RNA sequence (40-mer vRNA loop) is 5' - pGCA AAU CUU UCU CAC GUC CUG ACU UGU GAG UAA AAU UUG G - 3'. Due to the inability to precisely define bases, the exact nucleotide register remains ...Details: The RNA sequence (40-mer vRNA loop) is 5' - pGCA AAU CUU UCU CAC GUC CUG ACU UGU GAG UAA AAU UUG G - 3'. Due to the inability to precisely define bases, the exact nucleotide register remains elusive. Thus, in the modelling, generic purines (P5P) and generic pyrimidines (Y5P) were assigned based on apparent base size.
Source: (synth.) Tilapia lake virus
Has ligand of interestY
Has protein modificationN

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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: TiLV-NP bound to the 40-mer vRNA loop / Type: COMPLEX / Entity ID: all / 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / 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
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 366980 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028240
ELECTRON MICROSCOPYf_angle_d0.39711266
ELECTRON MICROSCOPYf_dihedral_angle_d11.6041464
ELECTRON MICROSCOPYf_chiral_restr0.0341287
ELECTRON MICROSCOPYf_plane_restr0.0031314

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