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- EMDB-5952: CryoEM reconstruction model of Orsay virus-like particle -

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Basic information

Entry
Database: EMDB / ID: EMD-5952
TitleCryoEM reconstruction model of Orsay virus-like particle
Map dataReconstruction of recombinant Orsay virus-like particle
Sample
  • Sample: recombinant virus-like particle of Orsay virus
  • Virus: Orsay virus
Keywordsvirus-like particle
Function / homologyNodavirus capsid / nodavirus capsid protein / Viral coat protein subunit / metal ion binding / Capsid protein alpha
Function and homology information
Biological speciesOrsay virus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsGuo YR / Hryc C / Jakana J / Jiang H / Wang D / Chiu W / Zhong W / Tao YJ
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Crystal structure of a nematode-infecting virus.
Authors: Yusong R Guo / Corey F Hryc / Joanita Jakana / Hongbing Jiang / David Wang / Wah Chiu / Weiwei Zhong / Yizhi J Tao /
Abstract: Orsay, the first virus discovered to naturally infect Caenorhabditis elegans or any nematode, has a bipartite, positive-sense RNA genome. Sequence analyses show that Orsay is related to nodaviruses, ...Orsay, the first virus discovered to naturally infect Caenorhabditis elegans or any nematode, has a bipartite, positive-sense RNA genome. Sequence analyses show that Orsay is related to nodaviruses, but molecular characterizations of Orsay reveal several unique features, such as the expression of a capsid-δ fusion protein and the use of an ATG-independent mechanism for translation initiation. Here we report the crystal structure of an Orsay virus-like particle assembled from recombinant capsid protein (CP). Orsay capsid has a T = 3 icosahedral symmetry with 60 trimeric surface spikes. Each CP can be divided into three regions: an N-terminal arm that forms an extended protein interaction network at the capsid interior, an S domain with a jelly-roll, β-barrel fold forming the continuous capsid, and a P domain that forms surface spike projections. The structure of the Orsay S domain is best aligned to T = 3 plant RNA viruses but exhibits substantial differences compared with the insect-infecting alphanodaviruses, which also lack the P domain in their CPs. The Orsay P domain is remotely related to the P1 domain in calicivirus and hepatitis E virus, suggesting a possible evolutionary relationship. Removing the N-terminal arm produced a slightly expanded capsid with fewer nucleic acids packaged, suggesting that the arm is important for capsid stability and genome packaging. Because C. elegans-Orsay serves as a highly tractable model for studying viral pathogenesis, our results should provide a valuable structural framework for further studies of Orsay replication and infection.
History
DepositionApr 25, 2014-
Header (metadata) releaseJul 30, 2014-
Map releaseAug 20, 2014-
UpdateSep 17, 2014-
Current statusSep 17, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5952.gif

Downloads & links

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Map

FileDownload / File: emd_5952.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of recombinant Orsay virus-like particle
Voxel sizeX=Y=Z: 2.15 Å
Density
Contour LevelBy AUTHOR: 1.6 / Movie #1: 1.6
Minimum - Maximum-5.35916138 - 9.55411816
Average (Standard dev.)0.23978099 (±0.90213323)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-97-101
Dimensions200200200
Spacing200200200
CellA=B=C: 430.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.152.152.15
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z430.000430.000430.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-97-100-101
NC/NR/NS200200200
D min/max/mean-5.3599.5540.240

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Supplemental data

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Sample components

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Entire : recombinant virus-like particle of Orsay virus

EntireName: recombinant virus-like particle of Orsay virus
Components
  • Sample: recombinant virus-like particle of Orsay virus
  • Virus: Orsay virus

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Supramolecule #1000: recombinant virus-like particle of Orsay virus

SupramoleculeName: recombinant virus-like particle of Orsay virus / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 7.7 MDa

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Supramolecule #1: Orsay virus

SupramoleculeName: Orsay virus / type: virus / ID: 1 / Name.synonym: Orsay virus-like particle / NCBI-ID: 977912 / Sci species name: Orsay virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No / Syn species name: Orsay virus-like particle
Host (natural)Organism: Caenorhabditis elegans (invertebrata) / synonym: INVERTEBRATES
Host systemOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta / Recombinant plasmid: pET28a
Molecular weightTheoretical: 7.2 MDa
Virus shellShell ID: 1 / Name: Orsay VLP Capsid Protein / Diameter: 360 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

Concentration4 mg/mL
BufferpH: 7.5
Details: 50 mM Tris, 500 mM NaCl, 2 mM EDTA, 5 mM 2-mercaptoethanol
GridDetails: 400 mesh Quantifoil R 1.2/1.3 copper grids with thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 80 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 25000
Specialist opticsEnergy filter - Name: in-column filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER
TemperatureAverage: 81 K
DateOct 2, 2012
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Digitization - Sampling interval: 6 µm / Number real images: 112 / Average electron dose: 25 e/Å2
Details: Each image is the average of subframes 2-36 after drift and damage correction.
Bits/pixel: 10

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Image processing

CTF correctionDetails: Particles per frame
Final two d classificationNumber classes: 25
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: OTHER / Software - Name: EMAN2
Details: The final map was generated from two independent data sets. Small subsets (~200 particles) of each data set were extracted from each half of the data and binned by 4X. Two independent ...Details: The final map was generated from two independent data sets. Small subsets (~200 particles) of each data set were extracted from each half of the data and binned by 4X. Two independent initial models were generated using EMAN2. The remaining particles were added to the two data sets and then refined independently (not binned) with icosahedral symmetry enforced. Angular step size was reduced after 15 iterations; after 25 iterations of refinement the data sets converged to a final density map. A low-pass filter was applied to the capsid protein portion of the density map and a soft-edge mask was used to remove the non-icosahedral portion of the density map (RNA). Gold Standard resolution assessment resulted in a final Fourier shell correlation (FSC) of 6.9A resolution between the two independent density maps, using the 0.143 criterion. The final density map was generated by combining the two data sets and performing one last round of refinement.
Number images used: 4332
Details112 images were imported into EMAN2, evaluated, and frames with a high amount of contamination and/or ice were removed. A total of 5,824 particles were boxed out (300 x 300 pixels) from the raw images using a semi-automated routine. Damage and drift correction done on the individual particles showed that minimal drift (<2A) had occurred for the complete data set. The corrected particles were imported back into EMAN2 and the contrast transfer function (CTF) was determined automatically followed by manual correction. Defocus was estimated to be between 1.3 and 4 um. Various subsets of particles were removed due on poor CTF parameters, resulting in the 4,332 particles that were used in the reconstruction.

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Atomic model buiding 1

Initial modelPDB ID:

4nwv

SoftwareName: Chimera
DetailsThe molecular model was determined by X-ray crystallography using this cryo-EM density map to phase the crystal. The model was then fit back into the density map with Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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