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- EMDB-6152: Structures of Protective Antibodies Reveal Sites of Vulnerability... -

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Entry
Database: EMDB / ID: EMD-6152
TitleStructures of Protective Antibodies Reveal Sites of Vulnerability on Ebola Virus
Map data
SampleFab fragment of c13C6 and c4G7 chimerized human monoclonal IgG1 antibody bound to Ebola virus glycoprotein
  • Ebola virus glycoprotein
  • (chimerized human IgG1 antigen binding fragment ...) x 2
KeywordsEbola / Antibody cocktails / mAbs / ZMAb / ZMapp / MB-003
Biological speciesEbola virus sp. / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 24 Å
AuthorsMurin CD / Fusco ML / Bornholdt ZA / Qiu X / Olinger GG / Zeitlin L / Kobinger GP / Ward AB / Saphire EO
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Structures of protective antibodies reveal sites of vulnerability on Ebola virus.
Authors: Charles D Murin / Marnie L Fusco / Zachary A Bornholdt / Xiangguo Qiu / Gene G Olinger / Larry Zeitlin / Gary P Kobinger / Andrew B Ward / Erica Ollmann Saphire /
Abstract: Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood ...Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.
History
DepositionOct 25, 2014-
Header (metadata) releaseNov 19, 2014-
Map releaseNov 19, 2014-
UpdateDec 10, 2014-
Current statusDec 10, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.65
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.65
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6152.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 80 pix.
= 328. Å
4.1 Å/pix.
x 80 pix.
= 328. Å
4.1 Å/pix.
x 80 pix.
= 328. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 3.65 / Movie #1: 3.65
Minimum - Maximum-6.99864483 - 11.645090100000001
Average (Standard dev.)0.00309111 (±0.99991232)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-6.99911.6450.003

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Supplemental data

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Sample components

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Entire Fab fragment of c13C6 and c4G7 chimerized human monoclonal IgG1 a...

EntireName: Fab fragment of c13C6 and c4G7 chimerized human monoclonal IgG1 antibody bound to Ebola virus glycoprotein
Number of Components: 3
Oligomeric State: Six Fab fragments bound to a single, trimeric GP (two Fabs per protomer)
MassTheoretical: 750 kDa

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Component #1: protein, Ebola virus glycoprotein

ProteinName: Ebola virus glycoprotein / a.k.a: EBOV GP / Oligomeric Details: trimer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 450 kDa
SourceSpecies: Ebola virus sp.
Source (engineered)Expression System: Drosophila melanogaster (fruit fly) / Vector: pMT-puro / Cell of expression system: Schneider 2 (S2)

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Component #2: protein, chimerized human IgG1 antigen binding fragment c13C6

ProteinName: chimerized human IgG1 antigen binding fragment c13C6 / a.k.a: c13C6 Fab / Oligomeric Details: monomer / Recombinant expression: Yes / Number of Copies: 3
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Nicotiana benthamiana (plant)

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Component #3: protein, chimerized human IgG1 antigen binding fragment c4G7

ProteinName: chimerized human IgG1 antigen binding fragment c4G7 / a.k.a: c4G7 Fab / Oligomeric Details: monomer / Number of Copies: 3 / Recombinant expression: Yes
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Nicotiana benthamiana (plant)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: negative staining
Sample solutionSpecimen conc.: 0.03 mg/mL / Buffer solution: 20 mM Tris, 150 mM NaCl / pH: 7.4
Support film400 Cu mesh
StainingTo prepare negative stain grids, a 4 uL aliquot of each complex, which had been diluted to a concentration of ~0.03 ug/mL with TBS buffer, was placed for 15 seconds onto carbon-coated 400 Cu mesh grids that had been plasma cleaned for 20 s (Gatan), blotted off on the edge of the grid, then immediately stained for 30 s with 4 uL of 2% uranyl formate. The stain was blotted off on the edge of the grid and the grid was allowed to dry.
VitrificationCryogen Name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Feb 5, 2014
Electron gunElectron Source: TUNGSTEN HAIRPIN / Accelerating Voltage: 120 kV / Illumination Mode: FLOOD BEAM
LensMagnification: 52000 X (nominal) / Imaging Mode: BRIGHT FIELD / Defocus: 1000 nm
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of Digital Images: 65

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C3 (3 fold cyclic) / Number of Projections: 10210
3D reconstructionSoftware: EMAN2, XMipp, IMAGIC / Resolution: 24 Å / Resolution Method: FSC 0.5, semi-independent

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