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

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Basic information

Entry
Database: PDB / ID: 6pci
TitleEBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 Fabs
Components
  • (Virion spike ...) x 2
  • (rEBOV-520 Fab ...) x 2
  • (rEBOV-548 Fab ...) x 2
Keywordsimmune system / viral protein / Ebola / filovirus / antibody / synergy
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 speciesEbola virus
Homo sapiens (human)
Zaire ebolavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.12 Å
AuthorsWard, A.B. / Murin, C.D. / Alkutkar, T.
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
DepositionJun 17, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / em_entity_assembly / 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 / 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 / _em_entity_assembly.entity_id_list / _pdbx_struct_assembly_gen.asym_id_list / _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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  • Superimposition on EM map
  • EMDB-20301
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Virion spike glycoprotein
H: rEBOV-548 Fab heavy chain
K: rEBOV-548 Fab light chain
D: Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (Makona) GP2
Q: rEBOV-520 Fab light chain
N: rEBOV-520 Fab heavy chain
B: Virion spike glycoprotein
I: rEBOV-548 Fab heavy chain
L: rEBOV-548 Fab light chain
E: Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (Makona) GP2
R: rEBOV-520 Fab light chain
O: rEBOV-520 Fab heavy chain
C: Virion spike glycoprotein
J: rEBOV-548 Fab heavy chain
M: rEBOV-548 Fab light chain
F: Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (Makona) GP2
S: rEBOV-520 Fab light chain
P: rEBOV-520 Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)453,27733
Polymers447,89018
Non-polymers5,38715
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Virion spike ... , 2 types, 6 molecules ABCDEF

#1: Protein Virion spike glycoprotein


Mass: 31096.926 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus / Gene: GP / Production host: Homo sapiens (human) / References: UniProt: A0A1C4HDV6, UniProt: A0A068J419*PLUS
#4: Protein Virion spike glycoprotein,Virion spike glycoprotein,Ebola Virus (Makona) GP2


Mass: 22142.592 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zaire ebolavirus, (gene. exp.) Ebola virus
Gene: GP / Production host: Homo sapiens (human) / References: UniProt: A0A0F6PDW0, UniProt: A0A068J419*PLUS

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Antibody , 4 types, 12 molecules HIJKLMQRSNOP

#2: Antibody rEBOV-548 Fab heavy chain


Mass: 25223.359 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody rEBOV-548 Fab light chain


Mass: 23324.775 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Antibody rEBOV-520 Fab light chain


Mass: 23434.941 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#6: Antibody rEBOV-520 Fab heavy chain


Mass: 24073.971 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Sugars , 2 types, 15 molecules

#7: 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
#8: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 12
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
1EBOV GPdMuc (Makona) in complex with rEBOV-520 and rEBOV-548 FabsCOMPLEX#1-#60MULTIPLE SOURCES
2Ebola Virus (Makona) GPCOMPLEX#1, #41MULTIPLE SOURCES
3rEBOV-548 FabCOMPLEX#2-#31MULTIPLE SOURCES
4rEBOV-520 FabCOMPLEX#5-#61MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11NO
210.16 MDaNO
310.45 MDaNO
410.45 MDaNO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.2
Details: Buffers were made fresh from 10X stocks. Detergent was made fresh in TBS as a 6X stock and added immediately prior to vitificaiton.
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlrodieNaCl1
220 mMtris(hydroxymethyl)aminomethaneTris1
30.01 % (w/v)Octyl Maltoside, FluorinatedF-OM1
SpecimenConc.: 4 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
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 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 / Nominal magnification: 29000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9.5 sec. / Electron dose: 51.85 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2832
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9cryoSPARC2initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13Rosettamodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13114 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5JQ3

5jq3

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