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- EMDB-8935: Ebola virus Makona variant GP (mucin-deleted) in complex with pan... -

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Basic information

Entry
Database: EMDB / ID: EMD-8935
TitleEbola virus Makona variant GP (mucin-deleted) in complex with pan-ebolavirus human antibody ADI-15878 Fab
Map dataEbola virus glycoprotein (mucin-deleted) Makona variant ectodomain bound to human survivor antibody ADI-15878 Fab.
Sample
  • Complex: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain bound to human survivor antibody ADI-15878 Fab
    • Complex: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain
      • Protein or peptide: Ebola virus Makona GP2
    • Complex: Human survivor antibody ADI-15878 Fab
      • Protein or peptide: Fv domain of ADI-15878 Fab light chain
      • Protein or peptide: Fv domain of ADI-15878 Fab heavy chain
  • Protein or peptide: Ebola virus Makona GP1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


clathrin-dependent endocytosis of virus by host cell / suppression by virus of host tetherin activity / entry receptor-mediated virion attachment to host cell / membrane => GO:0016020 / symbiont entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / lipid binding / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell plasma membrane ...clathrin-dependent endocytosis of virus by host cell / suppression by virus of host tetherin activity / entry receptor-mediated virion attachment to host cell / membrane => GO:0016020 / symbiont entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / lipid binding / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell plasma membrane / virion membrane / extracellular region / membrane
Similarity search - Function
Filoviruses glycoprotein, extracellular domain / Filoviruses glycoprotein / Filovirus glycoprotein
Similarity search - Domain/homology
Virion spike glycoprotein / Envelope glycoprotein
Similarity search - Component
Biological speciesEbola virus / Homo sa (virus) / Zaire ebolavirus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.14 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: Cell Rep / Year: 2018
Title: Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop.
Authors: Charles D Murin / Jessica F Bruhn / Zachary A Bornholdt / Jeffrey Copps / Robyn Stanfield / Andrew B Ward /
Abstract: Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The ...Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The antibody ADI-15878 was previously identified from a human survivor of Ebola virus Makona variant (EBOV/Mak) infection. This mAb demonstrated potent neutralizing activity against all known ebolaviruses and provided protection in rodent and ferret models against three ebolavirus species. Here, we describe the unliganded crystal structure of ADI-15878 as well as the cryo-EM structures of ADI-15878 in complex with the EBOV/Mak and Bundibugyo virus (BDBV) glycoproteins (GPs). ADI-15878 binds through an induced-fit mechanism by targeting highly conserved residues in the internal fusion loop (IFL), bridging across GP protomers via the heptad repeat 1 (HR1) region. Our structures provide a more complete description of the ebolavirus immunogenic landscape, as well as a molecular basis for how rare but potent antibodies target conserved filoviral fusion machinery.
History
DepositionJul 5, 2018-
Header (metadata) releaseJul 18, 2018-
Map releaseSep 12, 2018-
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: 0.0455
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0455
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6dzl
  • Surface level: 0.0455
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8935.png

Downloads & links

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Map

FileDownload / File: emd_8935.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEbola virus glycoprotein (mucin-deleted) Makona variant ectodomain bound to human survivor antibody ADI-15878 Fab.
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.0455 / Movie #1: 0.0455
Minimum - Maximum-0.035472646 - 0.11138579
Average (Standard dev.)0.00017306636 (±0.0024059904)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 460.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z460.000460.000460.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-69-50-137
NX/NY/NZ136114193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0350.1110.000

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Supplemental data

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Sample components

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Entire : Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) e...

EntireName: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain bound to human survivor antibody ADI-15878 Fab
Components
  • Complex: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain bound to human survivor antibody ADI-15878 Fab
    • Complex: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain
      • Protein or peptide: Ebola virus Makona GP2
    • Complex: Human survivor antibody ADI-15878 Fab
      • Protein or peptide: Fv domain of ADI-15878 Fab light chain
      • Protein or peptide: Fv domain of ADI-15878 Fab heavy chain
  • Protein or peptide: Ebola virus Makona GP1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) e...

SupramoleculeName: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain bound to human survivor antibody ADI-15878 Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#4
Source (natural)Organism: Ebola virus
Molecular weightTheoretical: 45 KDa

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Supramolecule #2: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) e...

SupramoleculeName: Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Ebola virus
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F / Recombinant plasmid: pPPI4

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Supramolecule #3: Human survivor antibody ADI-15878 Fab

SupramoleculeName: Human survivor antibody ADI-15878 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4 / Details: Fab was generated by papain digestion of IgG
Source (natural)Organism: Homo sa (virus)
Recombinant expressionOrganism: Nicotiana benthamiana (plant)

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Macromolecule #1: Ebola virus Makona GP1

MacromoleculeName: Ebola virus Makona GP1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Zaire ebolavirus / Strain: Kikwit-95
Molecular weightTheoretical: 35.51775 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 TAVNNNTHHQ DTGEESASSG KLGLITNTIA GVAGLITGGR RTR R

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Macromolecule #2: Ebola virus Makona GP2

MacromoleculeName: Ebola virus Makona GP2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Zaire ebolavirus / Strain: Kikwit-95
Molecular weightTheoretical: 18.334406 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVIVNAQPKC NPNLHFWTTQ DEGAAIGLAW IPYFGPAAEG IYTEGLMHNQ DGLICGLRQL ANETTQALQL FLRATTELRT FSILNRKAI DFLLQRWGGT CHILGPDCCI EPHDWTKNIT DKIDQIIHDV EVDDDDKAGW SHPQFEKGGG SGGGSGGGSW S HPQFEK

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Macromolecule #3: Fv domain of ADI-15878 Fab light chain

MacromoleculeName: Fv domain of ADI-15878 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.333459 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString:
DIVLTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GEAPKLLISD ASSLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQ QYYSSPTFGG GTKVEIK

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Macromolecule #4: Fv domain of ADI-15878 Fab heavy chain

MacromoleculeName: Fv domain of ADI-15878 Fab heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.171703 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString:
QVQLVQSGVT LVQPGGSLRV SCAASGFTFS SYAMSWVRQA PGKGLEWVSA ISGLGGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCAKD HRVWAAGYHF DYWGQGALVT VS

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / 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

Concentration1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 milimolarNaClSodium chloridesodium chloride
20.0 milimolarTrisTris

Details: Solutions were made fresh from 10X concentrate and filtered with a 0.2 micrometer filter into a sterile glass container.
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: 3.5s blot on both sides of the grid using Whattman No. 1 filter paper.
DetailsAntibody ADI-15878 Fab was added in 10 molar excess to glycoprotein and allowed to incubate overnight at 4 degrees Celsius followed by size exclusion chromatography to purify the complex and rid of excess Fab.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPreliminary grid screening was performed manually
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

8700


Details: Starting model was a negative stain reconstruction and low pass filtered
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Details: Particles were initially classified with 3D classification in Relion
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 157861
FSC plot (resolution estimation)

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