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- PDB-6uye: EBOV GPdMuc Makona bound to rEBOV-548 Fab -

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Basic information

Entry
Database: PDB / ID: 6uye
TitleEBOV GPdMuc Makona bound to rEBOV-548 Fab
Components
  • (Antibody rEBOV-548 Fab, ...) x 2
  • SGP
  • Virion spike glycoprotein
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / antibody / ebola / synergy / cross-reactive / therapeutic / VIRAL PROTEIN-IMMUNE SYSTEM complex
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsMurin, C.D. / Ward, A.B.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_validate_close_contact / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_validate_close_contact.auth_asym_id_2 / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: SGP
D: Virion spike glycoprotein
G: Antibody rEBOV-548 Fab, heavy chain
J: Antibody rEBOV-548 Fab, light chain
B: SGP
E: Virion spike glycoprotein
H: Antibody rEBOV-548 Fab, heavy chain
K: Antibody rEBOV-548 Fab, light chain
C: SGP
F: Virion spike glycoprotein
I: Antibody rEBOV-548 Fab, heavy chain
L: Antibody rEBOV-548 Fab, light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)206,39927
Polymers198,82012
Non-polymers7,57915
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44110 Å2
ΔGint-74 kcal/mol
Surface area77550 Å2

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Components

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Protein , 2 types, 6 molecules ABCDEF

#1: Protein SGP / glycoprotein GP1


Mass: 29058.561 Da / Num. of mol.: 3 / Fragment: UNP residues 32-292
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus / Strain: Makona / Gene: GP / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: A0A1C4HDL5, UniProt: A0A068J419*PLUS
#2: Protein Virion spike glycoprotein / glycoprotein GP2


Mass: 10798.289 Da / Num. of mol.: 3 / Fragment: UNP residues 503-598
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus / Strain: Makona / Gene: GP / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: A0A1C4HDV6, UniProt: A0A068J419*PLUS

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Antibody , 2 types, 6 molecules GHIJKL

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


Mass: 14680.421 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK 293F / Production host: Homo sapiens (human)
#4: Antibody Antibody rEBOV-548 Fab, light chain


Mass: 11736.019 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK 293F / Production host: Homo sapiens (human)

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Sugars , 4 types, 15 molecules

#5: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#6: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#7: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#8: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex of Ebola virus, mucin deleted, strain Makona in complex with rEBOV-548 FabCOMPLEX#1-#40MULTIPLE SOURCES
2Ebola virus, mucin deleted, strain MakonaCOMPLEX#1-#21RECOMBINANT
3rEBOV-548 FabCOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Ebola virus1570291Makona
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606HEK 293F
23Homo sapiens (human)9606HEK 293F
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
2200 mMTris1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47264 / Symmetry type: POINT

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