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- EMDB-0173: Complex of foot-and-mouth-disease virus (type O1 M) with the Fab ... -

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Basic information

Entry
Database: EMDB / ID: EMD-0173
TitleComplex of foot-and-mouth-disease virus (type O1 M) with the Fab of antibody D9. The virus is at 3.5 Ang resolution, the Fab is flexibly attached and at much lower resolution.
Map data
Sample
  • Virus: Foot-and-mouth disease virus in complex with D9 FAB
    • Complex: Foot-and-mouth disease virus
    • Complex: D9 FAB
Biological speciesFoot-and-mouth disease virus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.97 Å
AuthorsShimmon G / Kotecha A / Ren J / Asfor AS / Newman J / Berryman S / Cottam EM / Gold S / Tuthill TJ / King DP ...Shimmon G / Kotecha A / Ren J / Asfor AS / Newman J / Berryman S / Cottam EM / Gold S / Tuthill TJ / King DP / Brocchi E / King AMQ / Owens R / Fry EE / Stuart DI / Burman A / Jackson T
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)G1000099 United Kingdom
Wellcome Trust090532/Z/09/Z United Kingdom
Medical Research Council (United Kingdom)G1100525/1 United Kingdom
CitationJournal: PLoS One / Year: 2018
Title: Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays.
Authors: Gareth Shimmon / Abhay Kotecha / Jingshan Ren / Amin S Asfor / Joseph Newman / Stephen Berryman / Eleanor M Cottam / Sarah Gold / Toby J Tuthill / Donald P King / Emiliana Brocchi / Andrew M ...Authors: Gareth Shimmon / Abhay Kotecha / Jingshan Ren / Amin S Asfor / Joseph Newman / Stephen Berryman / Eleanor M Cottam / Sarah Gold / Toby J Tuthill / Donald P King / Emiliana Brocchi / Andrew M Q King / Ray Owens / Elizabeth E Fry / David I Stuart / Alison Burman / Terry Jackson /
Abstract: Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the ...Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.
History
DepositionAug 2, 2018-
Header (metadata) releaseAug 29, 2018-
Map releaseAug 29, 2018-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0425
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0425
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0173.png

Downloads & links

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Map

FileDownload / File: emd_0173.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 400 pix.
= 540. Å		1.35 Å/pix.
x 400 pix.
= 540. Å		1.35 Å/pix.
x 400 pix.
= 540. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0425 / Movie #1: 0.0425
Minimum - Maximum-0.10880451 - 0.18983106
Average (Standard dev.)0.0011324808 (±0.012099084)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0-3990
Dimensions400400400
Spacing400400400
CellA=B=C: 540.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z540.000540.000540.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-39900
NC/NR/NS400400400
D min/max/mean-0.1090.1900.001

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Supplemental data

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Additional map: This is unsharpened map where the density of...

Fileemd_0173_additional.map
AnnotationThis is unsharpened map where the density of flexible Fabs are visible.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Foot-and-mouth disease virus in complex with D9 FAB

EntireName: Foot-and-mouth disease virus in complex with D9 FAB
Components
  • Virus: Foot-and-mouth disease virus in complex with D9 FAB
    • Complex: Foot-and-mouth disease virus
    • Complex: D9 FAB

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Supramolecule #1: Foot-and-mouth disease virus in complex with D9 FAB

SupramoleculeName: Foot-and-mouth disease virus in complex with D9 FAB / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#6 / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 9 MDa
Virus shellShell ID: 1 / Name: Foot and Mouth Disease virus / Diameter: 300.0 Å / T number (triangulation number): 3

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Supramolecule #2: Foot-and-mouth disease virus

SupramoleculeName: Foot-and-mouth disease virus / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Foot-and-mouth disease virus
Recombinant expressionOrganism: Cricetinae gen. sp. (mammal)

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Supramolecule #3: D9 FAB

SupramoleculeName: D9 FAB / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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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 / Component - Concentration: 50.0 mM / Component - Name: HEPES
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 2-20 / Number grids imaged: 1 / Number real images: 360 / Average exposure time: 5.0 sec. / Average electron dose: 18.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 160000
Sample stageSpecimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.0.17)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1bbt

Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 1003
Initial angle assignmentType: COMMON LINE / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: COMMON LINE / Software - Name: RELION (ver. 1.3)
Final 3D classificationSoftware - Name: RELION (ver. 1.3)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 120 / Target criteria: Cross-correlation coefficient

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