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- EMDB-3246: Electron cryo-microscopy of Grapevine Fanleaf Virus complex with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3246
TitleElectron cryo-microscopy of Grapevine Fanleaf Virus complex with Nanobody
Map dataGrapevine Fanleaf Virus reconstruction
Sample
  • Sample: Grapevine Fanleaf Virus complex with Nanobodies
  • Virus: Grapevine fanleaf virus
  • Protein or peptide: Nanobody
Keywordsvirus / nanobody / complex
Function / homology
Function and homology information


transport of virus in host, cell to cell / host cell plasmodesma / viral capsid / host cell cytoplasm / host cell nucleus / structural molecule activity
Similarity search - Function
Nepovirus subgroup A, RNA2 polyprotein, Protein 2A / Nepovirus subgroup A polyprotein / Nepovirus coat protein / Nepovirus coat protein, C-terminal / Nepovirus coat protein, N-terminal / Nepovirus coat protein, central domain / Nepovirus coat protein, C-terminal domain / Nepovirus coat protein, N-terminal domain / Viral coat protein subunit
Similarity search - Domain/homology
Biological speciessynthetic construct (others) / Grapevine fanleaf virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHemmer C / Orlov I / Ackerer L / Marmonier A / Hleibieh K / Schmitt-Keichinger C / Vigne E / Gersch S / Komar V / Belval L ...Hemmer C / Orlov I / Ackerer L / Marmonier A / Hleibieh K / Schmitt-Keichinger C / Vigne E / Gersch S / Komar V / Belval L / Berthold F / Monsion B / Bron P / Lemaire O / Lorber B / Sgro JY / Gutierrez C / Muyldermans S / Demangeat G / Klaholz BP / Ritzenthaler C
CitationJournal: Plant Biotechnol J / Year: 2018
Title: Nanobody-mediated resistance to Grapevine fanleaf virus in plants.
Authors: Caroline Hemmer / Samia Djennane / Léa Ackerer / Kamal Hleibieh / Aurélie Marmonier / Sophie Gersch / Shahinez Garcia / Emmanuelle Vigne / Véronique Komar / Mireille Perrin / Claude Gertz ...Authors: Caroline Hemmer / Samia Djennane / Léa Ackerer / Kamal Hleibieh / Aurélie Marmonier / Sophie Gersch / Shahinez Garcia / Emmanuelle Vigne / Véronique Komar / Mireille Perrin / Claude Gertz / Lorène Belval / François Berthold / Baptiste Monsion / Corinne Schmitt-Keichinger / Olivier Lemaire / Bernard Lorber / Carlos Gutiérrez / Serge Muyldermans / Gérard Demangeat / Christophe Ritzenthaler /
Abstract: Since their discovery, single-domain antigen-binding fragments of camelid-derived heavy-chain-only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as ...Since their discovery, single-domain antigen-binding fragments of camelid-derived heavy-chain-only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode-transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell-to-cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.
History
DepositionNov 18, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseApr 13, 2016-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5foj
  • Surface level: 2.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5foj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3246.png

emd_3246_1.png

emd_3246_2.png

Downloads & links

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Map

FileDownload / File: emd_3246.map.gz / Format: CCP4 / Size: 210.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGrapevine Fanleaf Virus reconstruction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 384 pix.
= 409.344 Å		1.07 Å/pix.
x 384 pix.
= 409.344 Å		1.07 Å/pix.
x 384 pix.
= 409.344 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.066 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.67 / Movie #1: 2.2
Minimum - Maximum-9.91243362 - 13.45023823
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-192-192-192
Dimensions384384384
Spacing384384384
CellA=B=C: 409.344 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0661.0661.066
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z409.344409.344409.344
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS-192-192-192
NC/NR/NS384384384
D min/max/mean-9.91213.4500.000

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Supplemental data

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Sample components

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Entire : Grapevine Fanleaf Virus complex with Nanobodies

EntireName: Grapevine Fanleaf Virus complex with Nanobodies
Components
  • Sample: Grapevine Fanleaf Virus complex with Nanobodies
  • Virus: Grapevine fanleaf virus
  • Protein or peptide: Nanobody

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Supramolecule #1000: Grapevine Fanleaf Virus complex with Nanobodies

SupramoleculeName: Grapevine Fanleaf Virus complex with Nanobodies / type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Grapevine fanleaf virus

SupramoleculeName: Grapevine fanleaf virus / type: virus / ID: 1 / Details: Nanobodies were bound the virus surface. / NCBI-ID: 12274 / Sci species name: Grapevine fanleaf virus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: grapevine (unknown) / synonym: PLANTAE(HIGHER PLANTS)
Virus shellShell ID: 1 / T number (triangulation number): 3

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Macromolecule #1: Nanobody

MacromoleculeName: Nanobody / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: synthetic construct (others)

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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

Concentration0.5 mg/mL
BufferpH: 8.3 / Details: 100 mM Tris pH 8.3, 50 mM NaCl.
GridDetails: 300 mesh 2/2 Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III / Method: blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateDec 14, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1055 / Average electron dose: 50 e/Å2
Details: Every image is the average of seven frames recorded by the direct electron detector.
Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 120000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each Image
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Imagic / Number images used: 6139

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Atomic model buiding 1

Initial modelPDB ID:

4v5t

SoftwareName: Chimera, Coot
Detailsiterative manual model building (Coot) and structure refinement (Phenix) against the cryo-EM map.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5foj:
Cryo electron microscopy structure of Grapevine Fanleaf Virus complex with Nanobody

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