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- PDB-7tn9: Structure of the Inmazeb cocktail and resistance to escape agains... -

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Basic information

Entry
Database: PDB / ID: 7tn9
TitleStructure of the Inmazeb cocktail and resistance to escape against Ebola virus
Components
  • Envelope glycoprotein
  • GP2
  • REGN3470 heavy chain
  • REGN3470 light chain
  • REGN3471 heavy chain
  • REGN3471 light chain
  • REGN3479 heavy chain
  • REGN3479 light chain
KeywordsIMMUNE SYSTEM/VIRAL PROTEIN / Ebola / antiviral / therapeutics / IMMUNE SYSTEM-VIRAL PROTEIN complex
Function / homology
Function and homology information


host cell endoplasmic reticulum / viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / suppression by virus of host tetherin activity / host cell cytoplasm / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / membrane raft / fusion of virus membrane with host endosome membrane / viral envelope ...host cell endoplasmic reticulum / viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / suppression by virus of host tetherin activity / host cell cytoplasm / entry receptor-mediated virion attachment to host cell / symbiont entry into host cell / membrane raft / 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 / identical protein binding
Similarity search - Function
Filoviruses glycoprotein, extracellular domain / Filoviruses glycoprotein / Filovirus glycoprotein
Similarity search - Domain/homology
Envelope glycoprotein
Similarity search - Component
Biological speciesMus musculus (house mouse)
Ebola virus - Mayinga
Zaire
1976
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsRayaprolu, V. / Fulton, B. / Rafique, A. / Arturo, E. / Williams, D. / Hariharan, C. / Callaway, H. / Parvate, A. / Schendel, S.L. / Parekh, D. ...Rayaprolu, V. / Fulton, B. / Rafique, A. / Arturo, E. / Williams, D. / Hariharan, C. / Callaway, H. / Parvate, A. / Schendel, S.L. / Parekh, D. / Hui, S. / Shaffer, K. / Pascal, K.E. / Wloga, E. / Giordano, S. / Copin, R. / Franklin, M. / Boytz, R.M. / Donahue, C. / Davey, R. / Baum, A. / Kyratsous, C.A. / Saphire, E.O.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI142790 United States
Department of Health & Human Services (HHS)HHSO100201700016C United States
Department of Health & Human Services (HHS)HHSO100201700020C United States
CitationJournal: Cell Host Microbe / Year: 2023
Title: Structure of the Inmazeb cocktail and resistance to Ebola virus escape.
Authors: Vamseedhar Rayaprolu / Benjamin O Fulton / Ashique Rafique / Emilia Arturo / Dewight Williams / Chitra Hariharan / Heather Callaway / Amar Parvate / Sharon L Schendel / Diptiben Parekh / ...Authors: Vamseedhar Rayaprolu / Benjamin O Fulton / Ashique Rafique / Emilia Arturo / Dewight Williams / Chitra Hariharan / Heather Callaway / Amar Parvate / Sharon L Schendel / Diptiben Parekh / Sean Hui / Kelly Shaffer / Kristen E Pascal / Elzbieta Wloga / Stephanie Giordano / Nicole Negron / Min Ni / Richard Copin / Gurinder S Atwal / Matthew Franklin / Ruth Mabel Boytz / Callie Donahue / Robert Davey / Alina Baum / Christos A Kyratsous / Erica Ollmann Saphire /
Abstract: Monoclonal antibodies can provide important pre- or post-exposure protection against infectious disease for those not yet vaccinated or in individuals that fail to mount a protective immune response ...Monoclonal antibodies can provide important pre- or post-exposure protection against infectious disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, lessened disease and improved survival in a controlled trial. Here, we present the cryo-EM structure at 3.1 Å of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with the antibodies in the Inmazeb cocktail. This structure allows the modeling of previously disordered portions of the glycoprotein glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities and residues critical for resistance to escape by these and other clinically relevant antibodies. We further provide direct evidence that Inmazeb protects against the rapid emergence of escape mutants, whereas monotherapies even against conserved epitopes do not, supporting the benefit of a cocktail versus a monotherapy approach.
History
DepositionJan 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 22, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: REGN3471 heavy chain
T: Envelope glycoprotein
S: Envelope glycoprotein
U: Envelope glycoprotein
R: REGN3479 light chain
N: REGN3479 light chain
F: REGN3471 light chain
W: GP2
Q: REGN3479 heavy chain
M: REGN3479 heavy chain
G: REGN3470 heavy chain
H: REGN3470 light chain
P: REGN3479 light chain
O: REGN3479 heavy chain
V: GP2
X: GP2
I: REGN3470 heavy chain
J: REGN3470 light chain
C: REGN3471 heavy chain
E: REGN3471 heavy chain
B: REGN3471 light chain
D: REGN3471 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)532,35433
Polymers527,36522
Non-polymers4,98911
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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Protein , 2 types, 6 molecules TSUWVX

#2: Protein Envelope glycoprotein / GP1 / 2 / GP


Mass: 35605.875 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus - Mayinga, Zaire, 1976 / Strain: Mayinga-76 / Gene: GP / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q05320
#5: Protein GP2


Mass: 15795.782 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus - Mayinga, Zaire, 1976 / Strain: Mayinga-76 / Gene: GP / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q05320

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Antibody , 6 types, 16 molecules ACERNPFBDQMOGIHJ

#1: Antibody REGN3471 heavy chain


Mass: 23020.711 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody REGN3479 light chain


Mass: 23228.734 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#4: Antibody REGN3471 light chain


Mass: 24172.803 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#6: Antibody REGN3479 heavy chain


Mass: 22856.572 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#7: Antibody REGN3470 heavy chain


Mass: 23187.951 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)
#8: Antibody REGN3470 light chain


Mass: 23474.076 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Cricetulus griseus (Chinese hamster)

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Sugars , 2 types, 11 molecules

#9: Polysaccharide alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D- ...alpha-D-mannopyranose-(1-6)-alpha-D-mannopyranose-(1-6)-[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: 1072.964 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-6DManpa1-6[DManpa1-3]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-e1_e6-f1WURCSPDB2Glycan 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]{[(6+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#10: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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 interestY

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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
1Inmazeb therapeutic cocktailCOMPLEX#1, #3-#80MULTIPLE SOURCES
2Envelope glycoprotein, Shed GPCOMPLEX1RECOMBINANT
3REGN3471 heavy chain, REGN3479 light chain, REGN3471 light chain, REGN3479 heavy chain, REGN3470 heavy chain, REGN3470 light chainCOMPLEX1RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Ebola virus - Mayinga, Zaire, 1976128952
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Cricetulus griseus (Chinese hamster)10029
23Drosophila melanogaster (fruit fly)7227
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-baseC4H11NO31
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 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: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 52.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 6360
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC2.12particle selection
2SerialEMimage acquisition
4cryoSPARC2.12CTF correction
7Cootmodel fitting
12cryoSPARC2.123D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3154
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 276715 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5JQ3

5jq3

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00623158
ELECTRON MICROSCOPYf_angle_d0.81431437
ELECTRON MICROSCOPYf_dihedral_angle_d21.7573328
ELECTRON MICROSCOPYf_chiral_restr0.0523500
ELECTRON MICROSCOPYf_plane_restr0.0063986

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