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- PDB-7yr8: Lloviu cuevavirus nucleoprotein(1-450 residues)-RNA complex -

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Basic information

Entry
Database: PDB / ID: 7yr8
TitleLloviu cuevavirus nucleoprotein(1-450 residues)-RNA complex
Components
  • Nucleoprotein
  • RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')
KeywordsVIRAL PROTEIN / nucleoprotein
Function / homologyEbola nucleoprotein / Ebola nucleoprotein / viral RNA genome packaging / viral nucleocapsid / RNA / Nucleoprotein
Function and homology information
Biological speciesLloviu cuevavirus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHu, S.F. / Fujita-Fujiharu, Y. / Sugita, Y. / Wendt, L. / Muramoto, Y. / Nakano, M. / Hoenen, T. / Noda, T.
Funding support Japan, 6items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21J12207 Japan
Japan Society for the Promotion of Science (JSPS)21K07052 Japan
Japan Science and TechnologyJPMJFR214S Japan
Japan Agency for Medical Research and Development (AMED)21wm0325023j0002 Japan
Japan Science and TechnologyJPMJCR20HA Japan
Japan Agency for Medical Research and Development (AMED)JP22ama1214032 Japan
CitationJournal: PNAS Nexus / Year: 2023
Title: Cryoelectron microscopic structure of the nucleoprotein-RNA complex of the European filovirus, Lloviu virus.
Authors: Shangfan Hu / Yoko Fujita-Fujiharu / Yukihiko Sugita / Lisa Wendt / Yukiko Muramoto / Masahiro Nakano / Thomas Hoenen / Takeshi Noda /
Abstract: Lloviu virus (LLOV) is a novel filovirus detected in Schreiber's bats in Europe. The isolation of the infectious LLOV from bats has raised public health concerns. However, the virological and ...Lloviu virus (LLOV) is a novel filovirus detected in Schreiber's bats in Europe. The isolation of the infectious LLOV from bats has raised public health concerns. However, the virological and molecular characteristics of LLOV remain largely unknown. The nucleoprotein (NP) of LLOV encapsidates the viral genomic RNA to form a helical NP-RNA complex, which acts as a scaffold for nucleocapsid formation and de novo viral RNA synthesis. In this study, using single-particle cryoelectron microscopy, we determined two structures of the LLOV NP-RNA helical complex, comprising a full-length and a C-terminally truncated NP. The two helical structures were identical, demonstrating that the N-terminal region determines the helical arrangement of the NP. The LLOV NP-RNA protomers displayed a structure similar to that in the Ebola and Marburg virus, but the spatial arrangements in the helix differed. Structure-based mutational analysis identified amino acids involved in the helical assembly and viral RNA synthesis. These structures advance our understanding of the filovirus nucleocapsid formation and provide a structural basis for the development of antifiloviral therapeutics.
History
DepositionAug 9, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1May 17, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 24, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nucleoprotein
R: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')


Theoretical massNumber of molelcules
Total (without water)44,9902
Polymers44,9902
Non-polymers00
Water0
1
A: Nucleoprotein
R: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')
x 90


Theoretical massNumber of molelcules
Total (without water)4,049,067180
Polymers4,049,067180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation89
identity operation1_555x,y,z1
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 90 / Rise per n subunits: 3.16 Å / Rotation per n subunits: -15.11 °)

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Components

#1: Protein Nucleoprotein /


Mass: 43197.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lloviu cuevavirus / Strain: isolate Bat/Spain/Asturias-Bat86/2003 / Cell (production host): Adhension Cell Culture / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / Tissue (production host): Kidney(Embryonic) / References: UniProt: G8EFI1
#2: RNA chain RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')


Mass: 1792.037 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Lloviu cuevavirus nucleoprotein (1-450 residues) RNA complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Lloviu cuevavirus
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris(hydroxymethyl)aminomethaneTris-HClTris1
2150 mMSodium chlorideNaClSodium chloride1
325 mMMagnesium chlorideMgCl21
45 mMCalcium chlorideCaCl21
51 mMDithiothreitolDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: JEC-3000FC / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Apply 1.5 micro litters of sample from each side of the grid and blot for 6 seconds before plugging

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 150000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3780
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFIND4.1.14CTF correction
7Coot0.8.9.2model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 442067
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 399761 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 7YPW

7ypw


Pdb chain-ID: A / Accession code: 7YPW / Chain residue range: 20-406 / Pdb chain residue range: 20-406 / Source name: PDB / Type: experimental model

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