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- EMDB-3438: Three-dimensional reconstruction of W1118 strain Rosellinia necat... -

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Database: EMDB / ID: 3438
TitleThree-dimensional reconstruction of W1118 strain Rosellinia necatrix quadrivirus 1
Map dataReconstruction of Rosellinia necatrix quadrivirus 1 strain W1075
SampleRosellinia necatrix quadrivirus 1 empty particles W1118 strain:
virus
KeywordsdsRNA virus / quadrivirus / 3DR / fungal virus
SourceRosellinia necatrix quadrivirus 1 (RnQV1)
Methodsingle particle reconstruction / cryo EM / negative staining / 9.1 Å resolution
AuthorsLuque D / Mata CP / Gonzalez-Camacho F / Gonzalez JM / Gomez-Blanco J / Alfonso C / Rivas G / Havens WM / Kanematsu S / Suzuki N / Ghabrial SA / Trus BL / Caston JR
CitationJournal: J. Virol. / Year: 2016
Title: Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1.
Authors: Daniel Luque / Carlos P Mata / Fernando González-Camacho / José M González / Josué Gómez-Blanco / Carlos Alfonso / Germán Rivas / Wendy M Havens / Satoko Kanematsu / Nobuhiro Suzuki / Said A Ghabrial / Benes L Trus / José R Castón
Abstract: Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids ...Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization.
Importance: Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.
DateDeposition: May 19, 2016 / Header (metadata) release: Aug 10, 2016 / Map release: Aug 2, 2017 / Last update: Aug 2, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

Fileemd_3438.map.gz (map file in CCP4 format, 48779 KB)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
232 pix
2.54 Å/pix.
= 589.28 Å
232 pix
2.54 Å/pix.
= 589.28 Å
232 pix
2.54 Å/pix.
= 589.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.54 Å
Density
Contour Level:0.6 (by author), 0.6 (movie #1):
Minimum - Maximum-0.91213852 - 1.70096147
Average (Standard dev.)0.01264894 (0.23093329)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions232232232
Origin000
Limit231231231
Spacing232232232
CellA=B=C: 589.27997 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z232232232
origin x/y/z0.0000.0000.000
length x/y/z589.280589.280589.280
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS232232232
D min/max/mean-0.9121.7010.013

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Supplemental data

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Sample components

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Entire Rosellinia necatrix quadrivirus 1 empty particles W1118 strain

EntireName: Rosellinia necatrix quadrivirus 1 empty particles W1118 strain
Number of components: 1 / Oligomeric State: icosahedral
MassTheoretical: 15.6 MDa / Experimental: 15.9 MDa / Measured by: Sedimentation

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Component #1: virus, Rosellinia necatrix quadrivirus 1

VirusName: Rosellinia necatrix quadrivirus 1 / a.k.a: RnQV1 / Class: VIRION / Empty: Yes / Enveloped: No / Isolate: STRAIN
MassTheoretical: 15.6 MDa / Experimental: 15.9 MDa
SpeciesSpecies: Rosellinia necatrix quadrivirus 1 (RnQV1) / Strain: W1118
Source (natural)Host Species: Rosellinia necatrix / Host category: FUNGI / Host species strain: W1118

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining, cryo EM
Sample solutionBuffer solution: 50 mM Tris-HCl pH 7.8, 5 mM EDTA, 150 mM NaCl
pH: 7.8
Support filmR 2/2 Quantifoil grids
StainingSamples were applied to grids, blotted and plunged into liquid ethane
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Feb 18, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 10 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal), 50000 X (calibrated) / Cs: 2.26 mm / Imaging mode: BRIGHT FIELD / Defocus: 700 - 3800 nm
Specimen HolderHolder: Eucentric / Model: GATAN LIQUID NITROGEN
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 324 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 microns / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 23926
3D reconstructionAlgorithm: Projection Matching / Software: Xmipp / CTF correction: Phase flipping & amplitude decay / Resolution: 9.1 Å / Resolution method: FSC 0.333, semi-independent

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