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- EMDB-20301: EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs -

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Basic information

Entry
Database: EMDB / ID: EMD-20301
TitleEBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
Map dataEBOVGPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
Sample
  • Complex: EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
    • Complex: Ebola Virus (Makona) GP
      • Protein or peptide: Virion spike glycoprotein
      • Protein or peptide: Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (Makona) GP2
    • Complex: rEBOV-548 Fab
      • Protein or peptide: rEBOV-548 Fab heavy chain
      • Protein or peptide: rEBOV-548 Fab light chain
    • Complex: rEBOV-520 Fab
      • Protein or peptide: rEBOV-520 Fab light chain
      • Protein or peptide: rEBOV-520 Fab heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


membrane => GO:0016020 / viral envelope / extracellular region / membrane
Similarity search - Function
Filoviruses glycoprotein, extracellular domain / Filoviruses glycoprotein / Filovirus glycoprotein
Similarity search - Domain/homology
Envelope glycoprotein / Virion spike glycoprotein / Virion spike glycoprotein
Similarity search - Component
Biological speciesHomo sapiens (human) / Ebola virus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.12 Å
AuthorsWard AB / Murin CD / Alkutkar T
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
DepositionJun 17, 2019-
Header (metadata) releaseJun 26, 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: 0.0276
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0276
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pci
  • Surface level: 0.0276
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20301.png

Downloads & links

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Map

FileDownload / File: emd_20301.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEBOVGPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.0276 / Movie #1: 0.0276
Minimum - Maximum-0.05528564 - 0.099964745
Average (Standard dev.)0.00022900618 (±0.002622409)
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-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0550.1000.000

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Supplemental data

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Sample components

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Entire : EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs

EntireName: EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
Components
  • Complex: EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
    • Complex: Ebola Virus (Makona) GP
      • Protein or peptide: Virion spike glycoprotein
      • Protein or peptide: Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (Makona) GP2
    • Complex: rEBOV-548 Fab
      • Protein or peptide: rEBOV-548 Fab heavy chain
      • Protein or peptide: rEBOV-548 Fab light chain
    • Complex: rEBOV-520 Fab
      • Protein or peptide: rEBOV-520 Fab light chain
      • Protein or peptide: rEBOV-520 Fab heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs

SupramoleculeName: EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 450 KDa

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Supramolecule #2: Ebola Virus (Makona) GP

SupramoleculeName: Ebola Virus (Makona) GP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #4

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Supramolecule #3: rEBOV-548 Fab

SupramoleculeName: rEBOV-548 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

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Supramolecule #4: rEBOV-520 Fab

SupramoleculeName: rEBOV-520 Fab / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5-#6

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Macromolecule #1: Virion spike glycoprotein

MacromoleculeName: Virion spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus
Molecular weightTheoretical: 31.096926 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 AFWETKKNLT RKIRSEELSF T

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Macromolecule #2: rEBOV-548 Fab heavy chain

MacromoleculeName: rEBOV-548 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.223359 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQVEESGGG VVQPGGSLRL SCAASGFMFS NYGMHWVRQA PGKGLEWMAF IRYDDSKKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTALYYCAKE LLQVYTSAWG EGHSYYYALD VWGLGTAVTV SSASFKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV ...String:
QVQVEESGGG VVQPGGSLRL SCAASGFMFS NYGMHWVRQA PGKGLEWMAF IRYDDSKKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTALYYCAKE LLQVYTSAWG EGHSYYYALD VWGLGTAVTV SSASFKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSC

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Macromolecule #3: rEBOV-548 Fab light chain

MacromoleculeName: rEBOV-548 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.324775 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSPSS VSASVGDRVT ITCRASQDIS NWLAWYQQKP GKAPELLIYT ASILQSGVSS RFSGSGSGTD FTLTISSLQP EDSATYYCQ QGKSFPYTFG QGTKLEIKRT AAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQSPSS VSASVGDRVT ITCRASQDIS NWLAWYQQKP GKAPELLIYT ASILQSGVSS RFSGSGSGTD FTLTISSLQP EDSATYYCQ QGKSFPYTFG QGTKLEIKRT AAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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Macromolecule #4: Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (...

MacromoleculeName: Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (Makona) GP2
type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus
Molecular weightTheoretical: 22.142592 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: NNNTHHQDTG EESASSGKLG LITNTIAGVA GLITGGRRTR REVIVNAQPK CNPNLHFWTT QDEGAAIGLA WIPYFGPAAE GIYTEGLMH NQDGLICGLR QLANETTQAL QLFLRATTEL RTFSILNRKA IDFLLQRWGG TCHILGPDCC IEPHDWTKNI T DKIDQIIH ...String:
NNNTHHQDTG EESASSGKLG LITNTIAGVA GLITGGRRTR REVIVNAQPK CNPNLHFWTT QDEGAAIGLA WIPYFGPAAE GIYTEGLMH NQDGLICGLR QLANETTQAL QLFLRATTEL RTFSILNRKA IDFLLQRWGG TCHILGPDCC IEPHDWTKNI T DKIDQIIH DDDDKAGWSH PQFEKGGGSG GGSGGGSWSH PQFEK

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Macromolecule #5: rEBOV-520 Fab light chain

MacromoleculeName: rEBOV-520 Fab light chain / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.434941 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYFAWYQQK PGQAPRLLIS GTSTRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYFC QQYGNSLYTF GQGTKLEIKR TAAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYFAWYQQK PGQAPRLLIS GTSTRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYFC QQYGNSLYTF GQGTKLEIKR TAAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #6: rEBOV-520 Fab heavy chain

MacromoleculeName: rEBOV-520 Fab heavy chain / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.073971 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLQESGPG LGKPSETLSL SCTVSGASIR GYFWNWIRQP PGKGLEWIGY IHSTGSTNYN PSLKSRVTIS VDTSKNQVSL NVNSVTAAD TAVYFCARGA WNVATVYYYY GMDVWGQGTL VTVSSASFKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ...String:
QVQLQESGPG LGKPSETLSL SCTVSGASIR GYFWNWIRQP PGKGLEWIGY IHSTGSTNYN PSLKSRVTIS VDTSKNQVSL NVNSVTAAD TAVYFCARGA WNVATVYYYY GMDVWGQGTL VTVSSASFKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSC

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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: 12 / 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

Concentration4 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chlrodie
20.0 mMTristris(hydroxymethyl)aminomethane
0.01 % (w/v)F-OMOctyl Maltoside, Fluorinated

Details: Buffers were made fresh from 10X stocks. Detergent was made fresh in TBS as a 6X stock and added immediately prior to vitificaiton.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Sample was blotted on both sides of the grid. Sample equilibrated in the chamber for 10s before blotting for 5s..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2832 / Average exposure time: 9.5 sec. / Average electron dose: 51.85 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER
Details: Generated by ab initio reconstruction in CryoSPARC2
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 13114
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5jq3

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6pci:
EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs

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