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- PDB-6dzl: Ebola virus Makona variant GP (mucin-deleted) in complex with pan... -

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Basic information

Entry
Database: PDB / ID: 6dzl
TitleEbola virus Makona variant GP (mucin-deleted) in complex with pan-ebolavirus human antibody ADI-15878 Fab
Components
  • (Ebola virus Makona ...) x 2
  • (Fv domain of ADI-15878 Fab ...) x 2
KeywordsVIRAL PROTEIN / Ebola / antibody / pan-ebolavirus / internal fusion loop / HR1 / glycoprotein
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 speciesZaire ebolavirus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.14 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: 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, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 12, 2018Provider: repository / Type: Initial release
Revision 1.1Sep 19, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
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 / 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 / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _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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Assembly

Deposited unit
A: Ebola virus Makona GP1
D: Ebola virus Makona GP2
J: Fv domain of ADI-15878 Fab light chain
G: Fv domain of ADI-15878 Fab heavy chain
B: Ebola virus Makona GP1
E: Ebola virus Makona GP2
K: Fv domain of ADI-15878 Fab light chain
H: Fv domain of ADI-15878 Fab heavy chain
C: Ebola virus Makona GP1
F: Ebola virus Makona GP2
L: Fv domain of ADI-15878 Fab light chain
I: Fv domain of ADI-15878 Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)240,89124
Polymers235,07212
Non-polymers5,81912
Water0
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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Ebola virus Makona ... , 2 types, 6 molecules ABCDEF

#1: Protein Ebola virus Makona GP1


Mass: 35517.750 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zaire ebolavirus / Strain: Kikwit-95 / Gene: GP / Variant: Makona / Plasmid: pPPI4 / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: A0A0F7IM40, UniProt: P87666*PLUS
#2: Protein Ebola virus Makona GP2 / GP1 / 2 / GP


Mass: 18334.406 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zaire ebolavirus / Strain: Kikwit-95 / Gene: GP / Variant: Makona / Plasmid: pPPI4 / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: P87666

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Antibody , 2 types, 6 molecules JKLGHI

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


Mass: 11333.459 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Nicotiana benthamiana (plant)
#4: Antibody Fv domain of ADI-15878 Fab heavy chain


Mass: 13171.703 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Nicotiana benthamiana (plant)

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Sugars , 3 types, 12 molecules

#5: 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
#6: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D- ...alpha-D-mannopyranose-(1-2)-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: 1072.964 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


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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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-IDSourceDetails
1Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomain bound to human survivor antibody ADI-15878 FabCOMPLEX#2-#40MULTIPLE SOURCES
2Ebola virus Zaire (Makona variant) glycoprotein (mucin-deleted) ectodomainCOMPLEX#21RECOMBINANT
3Human survivor antibody ADI-15878 FabCOMPLEX#3-#41RECOMBINANTFab was generated by papain digestion of IgG
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.295 MDaNO
210.16 MDaNO
310.045 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Ebola virus1570291
32Ebola virus1570291
43Homo sa (virus)1570291
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Homo sapiens (human)9606HEK293FpPPI4
32Homo sapiens (human)9606HEK293FpPPI4
43Nicotiana benthamiana (plant)4100
Buffer solutionpH: 7.4
Details: Solutions were made fresh from 10X concentrate and filtered with a 0.2 micrometer filter into a sterile glass container.
Buffer component
IDConc.NameFormulaBuffer-ID
1150 milimolarsodium chlorideNaClSodium chloride1
220 milimolarTrisTris1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Antibody 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.
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: 3.5s blot on both sides of the grid using Whattman No. 1 filter paper

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA / Details: Preliminary grid screening was performed manually
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 43478 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
2RELION2.1particle selection
3Leginonimage acquisition
5GctfCTF correction
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 157861 / Symmetry type: POINT

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