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- EMDB-6151: Structures of Protective Antibodies Reveal Sites of Vulnerability... -

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Entry
Database: EMDB / ID: EMD-6151
TitleStructures of Protective Antibodies Reveal Sites of Vulnerability on Ebola Virus
Map dataReconstruction of Ebola virus glycoprotein in complex with Fab c2G4
Sample
  • Sample: Fab fragment of c2G4 chimerized human monoclonal IgG1 antibody bound to Ebola virus glycoprotein
  • Protein or peptide: Ebola virus glycoprotein
  • Protein or peptide: chimerized human IgG1 antigen binding fragment c2G4
KeywordsEbola / Antibody cocktails / mAbs / ZMAb / ZMapp / MB-003
Biological speciesEbola virus sp. / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 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: 5.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 5.11
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_6151.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Ebola virus glycoprotein in complex with Fab c2G4
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 5.11 / Movie #1: 5.11
Minimum - Maximum-5.66648054 - 16.038139340000001
Average (Standard dev.)0.00586917 (±0.99969113)
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-5.66616.0380.006

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Supplemental data

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Sample components

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Entire : Fab fragment of c2G4 chimerized human monoclonal IgG1 antibody bo...

EntireName: Fab fragment of c2G4 chimerized human monoclonal IgG1 antibody bound to Ebola virus glycoprotein
Components
  • Sample: Fab fragment of c2G4 chimerized human monoclonal IgG1 antibody bound to Ebola virus glycoprotein
  • Protein or peptide: Ebola virus glycoprotein
  • Protein or peptide: chimerized human IgG1 antigen binding fragment c2G4

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Supramolecule #1000: Fab fragment of c2G4 chimerized human monoclonal IgG1 antibody bo...

SupramoleculeName: Fab fragment of c2G4 chimerized human monoclonal IgG1 antibody bound to Ebola virus glycoprotein
type: sample / ID: 1000 / Oligomeric state: Three Fab fragments bound to a single GP / Number unique components: 2
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: Ebola virus glycoprotein

MacromoleculeName: Ebola virus glycoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: EBOV GP / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Ebola virus sp.
Molecular weightTheoretical: 450 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly) / Recombinant cell: Schneider 2 (S2) / Recombinant plasmid: pMT-puro

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Macromolecule #2: chimerized human IgG1 antigen binding fragment c2G4

MacromoleculeName: chimerized human IgG1 antigen binding fragment c2G4 / type: protein_or_peptide / ID: 2 / Name.synonym: c2G4 Fab / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Recombinant expressionOrganism: Nicotiana benthamiana (plant)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.03 mg/mL
BufferpH: 7.4 / Details: 20 mM Tris, 150 mM NaCl
StainingType: NEGATIVE
Details: To 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 ...Details: To 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.
GridDetails: 400 Cu mesh
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: OTHER
DateJan 15, 2014
Image recordingCategory: CCD / Film or detector model: OTHER / Number real images: 55
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: EMAN2, XMipp, IMAGIC / Number images used: 5772

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