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- EMDB-8818: Cryo-EM structure of the P73G mutant of cucumber necrosis virus u... -

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Database: EMDB / ID: 8818
TitleCryo-EM structure of the P73G mutant of cucumber necrosis virus under native conditions
Map dataCucumber necrosis virus, P73G mutant. Mutation blocks insect vector transmission but is buried in the capsid. Map shows disruption in the putative Zn binding site that may explain loss of transmission.
SampleCucumber necrosis virus:
virus
Function / homologyIcosahedral viral capsid protein, S domain / Viral coat protein subunit / Viral coat protein (S domain) / Plant viruses icosahedral capsid proteins 'S' region signature. / T=3 icosahedral viral capsid / structural molecule activity / RNA binding / Capsid protein
Function and homology information
SourceCucumber necrosis virus
Methodsingle particle reconstruction / cryo EM / 4.2 Å resolution
AuthorsSmith TJ
CitationJournal: J. Virol. / Year: 2017
Title: Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission.
Authors: Michael B Sherman / Kishore Kakani / D'Ann Rochon / Wen Jiang / Neil R Voss / Thomas J Smith
Abstract: (CNV) is a member of the genus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus As with other members of the genus, the CNV capsid swells ... (CNV) is a member of the genus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus As with other members of the genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the β-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the β-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the β-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission. (CNV), a member of the genus , is transmitted in nature via zoospores of the fungus While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the β-annulus at the icosahedral 3-fold axes. Therefore, the β-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.
DateDeposition: Jul 10, 2017 / Header (metadata) release: Jul 19, 2017 / Map release: Jul 19, 2017 / Last update: Sep 27, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

Fileemd_8818.map.gz (map file in CCP4 format, 665501 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
550 pix
1.03 Å/pix.
= 566.5 Å
550 pix
1.03 Å/pix.
= 566.5 Å
550 pix
1.03 Å/pix.
= 566.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density
Contour Level:0.5 (by author), 0.5 (movie #1):
Minimum - Maximum-3.9533381 - 2.8523855
Average (Standard dev.)-0.000000000048321 (0.16550784)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions550550550
Origin-275.0-275.0-275.0
Limit274.0274.0274.0
Spacing550550550
CellA=B=C: 566.5 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z550550550
origin x/y/z0.0000.0000.000
length x/y/z566.500566.500566.500
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-275-275-275
NC/NR/NS550550550
D min/max/mean-3.9532.852-0.000

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Supplemental data

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Sample components

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Entire Cucumber necrosis virus

EntireName: Cucumber necrosis virus / Number of components: 1

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Component #1: virus, Cucumber necrosis virus

VirusName: Cucumber necrosis virus / Class: VIRION / Empty: No / Enveloped: No / Isolate: STRAIN
SpeciesSpecies: Cucumber necrosis virus

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 37 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: DIRECT ELECTRON DE-20 (5k x 3k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 49525
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF

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