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- EMDB-6453: Electron cryo-microscopy of a betanodavirus virus-like particle -

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Basic information

Entry
Database: EMDB / ID: EMD-6453
TitleElectron cryo-microscopy of a betanodavirus virus-like particle
Map dataReconstruction of virus-like particle orange-spotted grouper nervous necrosis virus (OGNNV)
Sample
  • Sample: Virus-like particle of orange-spotted grouper nervous necrosis virus generated in E.coli
  • Virus: Orange-spotted grouper nervous necrosis virus
Keywordsvirus-like particle / orange-spotted grouper nervous necrosis virus / betanodavirus
Function / homologyNodavirus capsid / nodavirus capsid protein / Viral coat protein subunit / Virus-like particle of orange-spotted grouper nervous necrosis virus
Function and homology information
Biological speciesOrange-spotted grouper nervous necrosis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJunfeng XIE / Kunpeng LI / Yuanzhu GAO / Runqing HUANG / Yuxiong LAI / Yan SHI / Shaowei YANG / Guohua ZHU / Qinfen ZHANG / Jianguo HE
CitationJournal: Vet Res / Year: 2016
Title: Structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle.
Authors: Junfeng Xie / Kunpeng Li / Yuanzhu Gao / Runqing Huang / Yuxiong Lai / Yan Shi / Shaowei Yang / Guohua Zhu / Qinfen Zhang / Jianguo He /
Abstract: Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against ...Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development.
History
DepositionSep 3, 2015-
Header (metadata) releaseNov 18, 2015-
Map releaseOct 19, 2016-
UpdateOct 19, 2016-
Current statusOct 19, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbm
  • Surface level: 3.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jbm
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6453.gif

Downloads & links

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Map

FileDownload / File: emd_6453.map.gz / Format: CCP4 / Size: 412 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of virus-like particle orange-spotted grouper nervous necrosis virus (OGNNV)
Voxel sizeX=Y=Z: 0.933 Å
Density
Contour LevelBy AUTHOR: 3.2 / Movie #1: 3.2
Minimum - Maximum-9.71350861 - 14.21226978
Average (Standard dev.)0.00352491 (±0.94922048)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 447.84003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9330.9330.933
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z447.840447.840447.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-9.71414.2120.004

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Supplemental data

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Sample components

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Entire : Virus-like particle of orange-spotted grouper nervous necrosis vi...

EntireName: Virus-like particle of orange-spotted grouper nervous necrosis virus generated in E.coli
Components
  • Sample: Virus-like particle of orange-spotted grouper nervous necrosis virus generated in E.coli
  • Virus: Orange-spotted grouper nervous necrosis virus

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Supramolecule #1000: Virus-like particle of orange-spotted grouper nervous necrosis vi...

SupramoleculeName: Virus-like particle of orange-spotted grouper nervous necrosis virus generated in E.coli
type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 6.8 MDa

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Supramolecule #1: Orange-spotted grouper nervous necrosis virus

SupramoleculeName: Orange-spotted grouper nervous necrosis virus / type: virus / ID: 1 / NCBI-ID: 421750
Sci species name: Orange-spotted grouper nervous necrosis virus
Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Epinephelus coioides (orange-spotted grouper) / synonym: VERTEBRATES
Host systemOrganism: Escherichia coli (E. coli) / Recombinant strain: M15 / Recombinant plasmid: pQE30
Molecular weightTheoretical: 6.8 MDa
Virus shellShell ID: 1 / Diameter: 380 Å / T number (triangulation number): 3

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 8 / Details: PBS buffer
GridDetails: 300 mesh Quantifoil copper grid with thin carbon support, glow discharged in air atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK IV / Method: blot for 5 min before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.91 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 95 K / Max: 105 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200000 times magnification
DetailsWeak beam illumination
DateFeb 26, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 2.5 µm / Number real images: 1359 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: OTHER / Software - Name: EMAN, JSPR / Number images used: 32000
DetailsThe particles were selected manually.

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Atomic model buiding 1

Initial modelPDB ID:

4nww

SoftwareName: Coot, Phenix
DetailsLocal refinement
RefinementSpace: REAL
Output model

PDB-3jbm:
Electron cryo-microscopy of a virus-like particle of orange-spotted grouper nervous necrosis virus

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