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- EMDB-1870: Three-Dimensional Reconstruction of Heterocapsa circularisquama R... -

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Basic information

Entry
Database: EMDB / ID: EMD-1870
TitleThree-Dimensional Reconstruction of Heterocapsa circularisquama RNA Virus (HcRNAV-109) by Cryo-Electron Microscopy
Map dataHeterocapsa circularisquama ss RNA Virus (HcRNAV-109)
Sample
  • Sample: Heterocapsa circularisquama RNA virus (HcRNAV-109)
  • Virus: Heterocapsa circularisquama RNA virus
  • RNA: Singe Stranded RNA
KeywordsMarine Virus / single-stranded RNA Virus / handedness determination / Alvernaviridae / Dinornavirus
Biological speciesHeterocapsa circularisquama (eukaryote) / Heterocapsa circularisquama RNA virus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 18.0 Å
AuthorsMiller JL / Woodward JD / Chen S / Jaffer MA / Weber BW / Nagasaki K / Tomaru Y / Wepf R / Roseman A / Sewell BT / Varsani A
CitationJournal: J Gen Virol / Year: 2011
Title: Three-dimensional reconstruction of Heterocapsa circularisquama RNA virus by electron cryo-microscopy.
Authors: Jennifer L Miller / Jeremy Woodward / Shaoxia Chen / Mohammed Jaffer / Brandon Weber / Keizo Nagasaki / Yuji Tomaru / Roger Wepf / Alan Roseman / Arvind Varsani / Trevor Sewell /
Abstract: Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major ...Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T = 3 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a 'bump' on the surface of the protein. Though the capsid protein probably has the classic 'jelly roll' β-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection.
History
DepositionJan 31, 2011-
Header (metadata) releaseJun 23, 2011-
Map releaseJun 23, 2011-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1870.png

Downloads & links

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Map

FileDownload / File: emd_1870.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHeterocapsa circularisquama ss RNA Virus (HcRNAV-109)
Voxel sizeX=Y=Z: 3.616 Å
Density
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.0394518 - 0.0778067
Average (Standard dev.)-0.000481025 (±0.00622063)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 723.2 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.6163.6163.616
M x/y/z200200200
origin x/y/z-0.000-0.000-0.000
length x/y/z723.200723.200723.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-160-160-159
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.0390.078-0.000

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Supplemental data

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Sample components

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Entire : Heterocapsa circularisquama RNA virus (HcRNAV-109)

EntireName: Heterocapsa circularisquama RNA virus (HcRNAV-109)
Components
  • Sample: Heterocapsa circularisquama RNA virus (HcRNAV-109)
  • Virus: Heterocapsa circularisquama RNA virus
  • RNA: Singe Stranded RNA

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Supramolecule #1000: Heterocapsa circularisquama RNA virus (HcRNAV-109)

SupramoleculeName: Heterocapsa circularisquama RNA virus (HcRNAV-109) / type: sample / ID: 1000 / Details: T equals 3
Oligomeric state: Virus particle containing 180 identical protein subunits and one single-stranded RNA molecule
Number unique components: 2

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Supramolecule #1: Heterocapsa circularisquama RNA virus

SupramoleculeName: Heterocapsa circularisquama RNA virus / type: virus / ID: 1 / Name.synonym: HcRNAV-109 / NCBI-ID: 331975 / Sci species name: Heterocapsa circularisquama RNA virus / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No / Syn species name: HcRNAV-109
Host (natural)Organism: Heterocapsa circularisquama (eukaryote) / synonym: ALGAE
Virus shellShell ID: 1 / Diameter: 340 Å / T number (triangulation number): 3

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Macromolecule #1: Singe Stranded RNA

MacromoleculeName: Singe Stranded RNA / type: rna / ID: 1 / Name.synonym: Singe Stranded RNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Heterocapsa circularisquama (eukaryote)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 10 mM Na2HPO4 and 10 mM KH2PO4 in distilled water
StainingType: NEGATIVE / Details: Cryo in ice
GridDetails: 300 mesh copper R2/2 Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Home-made plunger
Method: Blot 3 seconds, re-apply virus, blot 2 seconds. plunge

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 55299.5 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.8 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Side entry cryo holder Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 1.808 µm / Number real images: 108 / Average electron dose: 10 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final angle assignmentDetails: SPIDER
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MRC, EMAN, SPIDER
Details: We used two different starting models for refinement - one generated by EMAN starticos and one generated by applying the orientations determined by MRC refine
Number images used: 2593
DetailsThe concentration of virus in the sample was very low

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