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- EMDB-20947: EBOV GPdMuc Makona bound to rEBOV-548 Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-20947
TitleEBOV GPdMuc Makona bound to rEBOV-548 Fab
Map dataEbola virus glycoprotein mucin deleted in complex with antibody Fab rEBOV-548
Sample
  • Complex: Complex of Ebola virus, mucin deleted, strain Makona in complex with rEBOV-548 Fab
    • Complex: Ebola virus, mucin deleted, strain MakonaZaire ebolavirus
      • Protein or peptide: SGP
      • Protein or peptide: Virion spike glycoprotein
    • Complex: rEBOV-548 Fab
      • Protein or peptide: Antibody rEBOV-548 Fab, heavy chain
      • Protein or peptide: Antibody rEBOV-548 Fab, light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homologyFiloviruses glycoprotein, extracellular domain / Filoviruses glycoprotein / Filovirus glycoprotein / viral envelope / extracellular region / membrane / Envelope glycoprotein / SGP / Virion spike glycoprotein
Function and homology information
Biological speciesEbola virus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsMurin CD / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI109762 United States
CitationJournal: Immunity / Year: 2020
Title: Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.
Authors: Pavlo Gilchuk / Charles D Murin / Jacob C Milligan / Robert W Cross / Chad E Mire / Philipp A Ilinykh / Kai Huang / Natalia Kuzmina / Pilar X Altman / Sean Hui / Bronwyn M Gunn / Aubrey L ...Authors: Pavlo Gilchuk / Charles D Murin / Jacob C Milligan / Robert W Cross / Chad E Mire / Philipp A Ilinykh / Kai Huang / Natalia Kuzmina / Pilar X Altman / Sean Hui / Bronwyn M Gunn / Aubrey L Bryan / Edgar Davidson / Benjamin J Doranz / Hannah L Turner / Tanwee Alkutkar / Robin Flinko / Chiara Orlandi / Robert Carnahan / Rachel Nargi / Robin G Bombardi / Megan E Vodzak / Sheng Li / Adaora Okoli / Morris Ibeawuchi / Benjamin Ohiaeri / George K Lewis / Galit Alter / Alexander Bukreyev / Erica Ollmann Saphire / Thomas W Geisbert / Andrew B Ward / James E Crowe /
Abstract: Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb ...Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.
History
DepositionNov 13, 2019-
Header (metadata) releaseNov 20, 2019-
Map releaseMar 11, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uye
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6uye
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20947.png

Downloads & links

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Map

FileDownload / File: emd_20947.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEbola virus glycoprotein mucin deleted in complex with antibody Fab rEBOV-548
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.7769421 - 2.8894129
Average (Standard dev.)-0.00011447525 (±0.07421249)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 296.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z296.640296.640296.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-1.7772.889-0.000

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Supplemental data

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Sample components

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Entire : Complex of Ebola virus, mucin deleted, strain Makona in complex w...

EntireName: Complex of Ebola virus, mucin deleted, strain Makona in complex with rEBOV-548 Fab
Components
  • Complex: Complex of Ebola virus, mucin deleted, strain Makona in complex with rEBOV-548 Fab
    • Complex: Ebola virus, mucin deleted, strain MakonaZaire ebolavirus
      • Protein or peptide: SGP
      • Protein or peptide: Virion spike glycoprotein
    • Complex: rEBOV-548 Fab
      • Protein or peptide: Antibody rEBOV-548 Fab, heavy chain
      • Protein or peptide: Antibody rEBOV-548 Fab, light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Complex of Ebola virus, mucin deleted, strain Makona in complex w...

SupramoleculeName: Complex of Ebola virus, mucin deleted, strain Makona in complex with rEBOV-548 Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: Ebola virus, mucin deleted, strain Makona

SupramoleculeName: Ebola virus, mucin deleted, strain Makona / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Ebola virus / Strain: Makona
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F

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Supramolecule #3: rEBOV-548 Fab

SupramoleculeName: rEBOV-548 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F

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Macromolecule #1: SGP

MacromoleculeName: SGP / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus / Strain: Makona
Molecular weightTheoretical: 29.058561 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SIPLGVIHNS TLQVSDVDKL VCRDKLSSTN QLRSVGLNLE GNGVATDVPS VTKRWGFRSG VPPKVVNYEA GEWAENCYNL EIKKPDGSE CLPAAPDGIR GFPRCRYVHK VSGTGPCAGD FAFHKEGAFF LYDRLASTVI YRGTTFAEGV VAFLILPQAK K DFFSSHPL ...String:
SIPLGVIHNS TLQVSDVDKL VCRDKLSSTN QLRSVGLNLE GNGVATDVPS VTKRWGFRSG VPPKVVNYEA GEWAENCYNL EIKKPDGSE CLPAAPDGIR GFPRCRYVHK VSGTGPCAGD FAFHKEGAFF LYDRLASTVI YRGTTFAEGV VAFLILPQAK K DFFSSHPL REPVNATEDP SSGYYSTTIR YQATGFGTNE TEYLFEVDNL TYVQLESRFT PQFLLQLNET IYASGKRSNT TG KLIWKVN PEIDTTIGEW AFWE

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Macromolecule #2: Virion spike glycoprotein

MacromoleculeName: Virion spike glycoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus / Strain: Makona
Molecular weightTheoretical: 10.798289 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
VIVNAQPKCN PNLHYWTTQD EGAAIGLAWI PYFGPAAEGI YTEGLMHNQD GLICGLRQLA NETTQALQLF LRATTELRTF SILNRKAID FLLQRWG

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Macromolecule #3: Antibody rEBOV-548 Fab, heavy chain

MacromoleculeName: Antibody rEBOV-548 Fab, heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.680421 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QVQVEESGGG VVQPGGSLRL SCAASGFMFS NYGMHWVRQA PGKGLEWMAF IRYDDSKKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTALYYCAKE LLQVYTSAWG EGHSYYYALD VWGLGTAVTV SSA

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Macromolecule #4: Antibody rEBOV-548 Fab, light chain

MacromoleculeName: Antibody rEBOV-548 Fab, light chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.736019 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
IQMTQSPSSV SASVGDRVTI TCRASQDISN WLAWYQQKPG KAPELLIYTA SILQSGVSSR FSGSGSGTDF TLTISSLQPE DSATYYCQQ GKSFPYTFGQ GTKLEIKRT

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration3.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
200.0 mMTris
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.0) / Number images used: 47264
FSC plot (resolution estimation)

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