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- EMDB-34016: Lloviu cuevavirus nucleoprotein-RNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-34016
TitleLloviu cuevavirus nucleoprotein-RNA complex
Map data
Sample
  • Complex: Lloviu cuevavirus nucleoprotein RNA complex
    • Protein or peptide: Nucleoprotein
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')
Function / homologyEbola nucleoprotein / Ebola nucleoprotein / viral RNA genome packaging / viral nucleocapsid / Nucleoprotein
Function and homology information
Biological speciesLloviu cuevavirus / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0356 Å
AuthorsHu SF / Fujita-Fujiharu Y / Sugita Y / Wendt L / Muramoto Y / Nakano M / Hoenen T / Noda T
Funding support Japan, 6 items
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 4, 2022-
Header (metadata) releaseApr 19, 2023-
Map releaseApr 19, 2023-
UpdateMay 24, 2023-
Current statusMay 24, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34016.png

Downloads & links

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Map

FileDownload / File: emd_34016.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.13 Å
Density
Contour LevelBy AUTHOR: 0.045
Minimum - Maximum-0.17110413 - 0.2948582
Average (Standard dev.)0.0005879972 (±0.014132421)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 452.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34016_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34016_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lloviu cuevavirus nucleoprotein RNA complex

EntireName: Lloviu cuevavirus nucleoprotein RNA complex
Components
  • Complex: Lloviu cuevavirus nucleoprotein RNA complex
    • Protein or peptide: Nucleoprotein
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')

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Supramolecule #1: Lloviu cuevavirus nucleoprotein RNA complex

SupramoleculeName: Lloviu cuevavirus nucleoprotein RNA complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lloviu cuevavirus

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Macromolecule #1: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lloviu cuevavirus / Strain: isolate Bat/Spain/Asturias-Bat86/2003
Molecular weightTheoretical: 84.553094 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNRYLGHGTR TSRENTNLSE LHGILSLGLN VDHTIVRKKS IPLFEIGNSD QVCNWIIQII EAGVDLQEVA DSFLTMLCVN HAYQGDPNL FLESPAAHYL KGHGIHFEIQ HRDNVDHITD LLGVGSRDKS LRKTLSALEF EPGGTTTAGM FLSFASLFLP K LVVGERAC ...String:
MNRYLGHGTR TSRENTNLSE LHGILSLGLN VDHTIVRKKS IPLFEIGNSD QVCNWIIQII EAGVDLQEVA DSFLTMLCVN HAYQGDPNL FLESPAAHYL KGHGIHFEIQ HRDNVDHITD LLGVGSRDKS LRKTLSALEF EPGGTTTAGM FLSFASLFLP K LVVGERAC LEKVQRQIQI HAEQGLIQYP TQWQSVGHMM VVFRLIRVNF VLKFLLVHQG MHMMAGHDAN DAIIANSISQ TR FSGLLIV KTVLEHILQK TEAGVQLHPL ARTSKVKGEL LAFKSALEAL ASHREYAPFA RLLNLSGVNN LEHGLYPQLS AIA LGVATA HGSTLAGVNV SEQYQQLREA ATEAEKQLQQ HSEMRELETL GLDEQERKIL ATFHSRKNEI NIQQTSSILA IRKE RLRKL TEALTEEKNK NALDDEDESE EDDWSPENRG IRSNKGSTKE PSSYTASRTE EDRNNYNSKD DHLSGKEQMS TQQES GADD LDLFDLDDDG DTNSQDPNNR QKQSDTQQTQ ESSDRSDYSR RPAYDWPPGD RPHTTQATDE HTDLLNKDHR RNQVKP GRR GNDPRTLPLI SFDDNEGEIL DDKSDLPAPD THSDPDTEES EEEHPDEELL PPAPKYSTKT SEQEPGDWKQ PTSPLST IP EEEGGHEANN DNSESDLIDQ MYQHIFKTEG AYAAINYYYK TTGRPVTFTS NNNHDYTFPQ DTEGLFPPWE GKENQKVA E ILTNSLHETG QEWADMSAKE RYLFLINN

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Macromolecule #2: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')

MacromoleculeName: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.792037 KDa
SequenceString:
UUUUUU

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMTris-HClTrisTris(hydroxymethyl)aminomethane Hydrochloride
150.0 mMNaClSodium chloridesodium chloride
25.0 mMMgCl2magnesium chloride
5.0 mMCaCl2calcium chloride
1.0 mMDTTdithiothreitol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Apply 1.25 micro litters of sample from each side of the grid and blot for 7 seconds before plugging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector with two hexapole elements
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 3122 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 62855
Startup modelType of model: OTHER / Details: cylinder created by relion_helix_toolbox
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: RELION 3D classification
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0356 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 26797
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5z9w


Chain - Chain ID: A / Chain - Residue range: 20-400
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7ypw:
Lloviu cuevavirus nucleoprotein-RNA complex

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