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- EMDB-8227: Tomographic subvolume average of membrane-bound Ebola (EBOV-Makon... -

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Entry
Database: EMDB / ID: EMD-8227
TitleTomographic subvolume average of membrane-bound Ebola (EBOV-Makona) glycoprotein bound to c2G4 antibody
Map dataMembrane-bound Ebola (EBOV-Makona) glycoprotein bound to c2G4 antibody
Samplevirus-like particle / antibody complex != Ebola virus sp.

virus-like particle / antibody complex

  • Complex: virus-like particle / antibody complex
    • Virus: Ebola virus sp.
    • Complex: c2G4 antibody
Biological speciesHomo sapiens (human) / Ebola virus sp.
Methodsubtomogram averaging / cryo EM / Resolution: 25.0 Å
AuthorsTran EEH / Subramaniam S
CitationJournal: J Virol / Year: 2016
Title: Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry.
Authors: Erin E H Tran / Elizabeth A Nelson / Pranay Bonagiri / James A Simmons / Charles J Shoemaker / Connie S Schmaljohn / Gary P Kobinger / Larry Zeitlin / Sriram Subramaniam / Judith M White /
Abstract: ZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West ...ZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1(+) endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env.
IMPORTANCE: The recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar ...IMPORTANCE: The recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed-embedded in the membrane and present at high density-on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.
History
DepositionJun 3, 2016-
Header (metadata) releaseJun 15, 2016-
Map releaseJun 15, 2016-
UpdateApr 11, 2018-
Current statusApr 11, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.494
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.494
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8227.png

Downloads & links

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Map

FileDownload / File: emd_8227.map.gz / Format: CCP4 / Size: 1.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMembrane-bound Ebola (EBOV-Makona) glycoprotein bound to c2G4 antibody
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 99 pix.
= 405.9 Å		4.1 Å/pix.
x 55 pix.
= 225.5 Å		4.1 Å/pix.
x 55 pix.
= 225.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 4.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.494 / Movie #1: 0.494
Minimum - Maximum-3.4439044 - 4.17309
Average (Standard dev.)-0.012896408 (±0.545072)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions555599
Spacing555599
CellA: 225.5 Å / B: 225.5 Å / C: 405.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z555599
origin x/y/z0.0000.0000.000
length x/y/z225.500225.500405.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS555599
D min/max/mean-3.4444.173-0.013

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Supplemental data

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Sample components

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Entire : virus-like particle / antibody complex

EntireName: virus-like particle / antibody complex
Components
  • Complex: virus-like particle / antibody complex
    • Virus: Ebola virus sp.
    • Complex: c2G4 antibody

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Supramolecule #2: Ebola virus sp.

SupramoleculeName: Ebola virus sp. / type: virus / ID: 2 / Parent: 1
Details: 293T/17 cells were transfected with cDNAs encoding EBOV GP, VP40, mCherry-tagged VP40, and beta-lactamase-tagged VP40. After transfection, VLPs were purified from the cell medium by ...Details: 293T/17 cells were transfected with cDNAs encoding EBOV GP, VP40, mCherry-tagged VP40, and beta-lactamase-tagged VP40. After transfection, VLPs were purified from the cell medium by centrifugation through a sucrose cushion.
NCBI-ID: 205488 / Sci species name: Ebola virus sp. / Sci species strain: EBOV-Makona VLP / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: Yes
Host systemOrganism: Homo sapiens (human) / Recombinant cell: HEK 293T/17 / Recombinant plasmid: pWRG7077

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Supramolecule #1: virus-like particle / antibody complex

SupramoleculeName: virus-like particle / antibody complex / type: complex / ID: 1 / Parent: 0

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Supramolecule #3: c2G4 antibody

SupramoleculeName: c2G4 antibody / type: complex / ID: 3 / Parent: 1
Details: Whole chimerized human IgG from ZMapp antibody cocktail that binds the base region of Ebola virus glycoproteins
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.4
Details: 10% sucrose-HM (20 mM HEPES, 20 mM MES, 130 mM NaCl)
GridModel: Quantifoil Multi-A / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 294 K / Instrument: LEICA EM GP
Details: Blot for 6 seconds before plunging into liquid ethane (LEICA EM GP)..
Details8 micrograms of Ebola VLPs were incubated with 12.5 micrograms of c2G4 antibody on ice for ~30-60 minutes before plunge-freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: Gatan, Inc / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 0-4 / Number grids imaged: 2 / Average exposure time: 2.0 sec. / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 23000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: OTHER
Details: The average resolution of all maps was estimated based on comparison with features observed when X-ray structures are filtered to this resolution.
Number subtomograms used: 1998
ExtractionNumber tomograms: 26 / Number images used: 2470 / Method: Volumes were picked automatically.
Final angle assignmentType: OTHER

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Target criteria: Fitting was based on location of antibody escape mutant residues.

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