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- EMDB-26005: Structure of the Inmazeb cocktail and resistance to escape agains... -

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Basic information

Entry
Database: EMDB / ID: EMD-26005
TitleStructure of the Inmazeb cocktail and resistance to escape against Ebola virus
Map dataInmazeb cocktail
Sample
  • Complex: Inmazeb therapeutic cocktail
    • Complex: Envelope glycoprotein, Shed GP
    • Complex: REGN3471 heavy chain, REGN3479 light chain, REGN3471 light chain, REGN3479 heavy chain, REGN3470 heavy chain, REGN3470 light chain
    • Protein or peptide: REGN3471 heavy chain
    • Protein or peptide: REGN3479 light chain
    • Protein or peptide: REGN3471 light chain
    • Protein or peptide: GP2
    • Protein or peptide: REGN3479 heavy chain
    • Protein or peptide: REGN3470 heavy chain
    • Protein or peptide: REGN3470 light chain
  • Protein or peptide: Envelope glycoprotein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 / 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
Methodsingle 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 SL / Parekh D ...Rayaprolu V / Fulton B / Rafique A / Arturo E / Williams D / Hariharan C / Callaway H / Parvate A / Schendel SL / Parekh D / Hui S / Shaffer K / Pascal KE / Wloga E / Giordano S / Copin R / Franklin M / Boytz RM / Donahue C / Davey R / Baum A / Kyratsous CA / Saphire EO
Funding support United States, 3 items
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, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateFeb 22, 2023-
Current statusFeb 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26005.png

Downloads & links

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Map

FileDownload / File: emd_26005.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInmazeb cocktail
Voxel sizeX=Y=Z: 1.0504 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.6188447 - 1.717513
Average (Standard dev.)-0.00014677132 (±0.029737614)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 537.8048 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Inmazeb cocktail

Fileemd_26005_half_map_1.map
AnnotationInmazeb cocktail
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Inmazeb cocktail

Fileemd_26005_half_map_2.map
AnnotationInmazeb cocktail
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Inmazeb therapeutic cocktail

EntireName: Inmazeb therapeutic cocktail
Components
  • Complex: Inmazeb therapeutic cocktail
    • Complex: Envelope glycoprotein, Shed GP
    • Complex: REGN3471 heavy chain, REGN3479 light chain, REGN3471 light chain, REGN3479 heavy chain, REGN3470 heavy chain, REGN3470 light chain
    • Protein or peptide: REGN3471 heavy chain
    • Protein or peptide: REGN3479 light chain
    • Protein or peptide: REGN3471 light chain
    • Protein or peptide: GP2
    • Protein or peptide: REGN3479 heavy chain
    • Protein or peptide: REGN3470 heavy chain
    • Protein or peptide: REGN3470 light chain
  • Protein or peptide: Envelope glycoprotein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Inmazeb therapeutic cocktail

SupramoleculeName: Inmazeb therapeutic cocktail / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1, #3-#8

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Supramolecule #2: Envelope glycoprotein, Shed GP

SupramoleculeName: Envelope glycoprotein, Shed GP / type: complex / ID: 2 / Chimera: Yes / Parent: 1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: REGN3471 heavy chain, REGN3479 light chain, REGN3471 light chain,...

SupramoleculeName: REGN3471 heavy chain, REGN3479 light chain, REGN3471 light chain, REGN3479 heavy chain, REGN3470 heavy chain, REGN3470 light chain
type: complex / ID: 3 / Chimera: Yes / Parent: 1
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976

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Macromolecule #1: REGN3471 heavy chain

MacromoleculeName: REGN3471 heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.020711 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYDMHWVRQA TGKGLEWVSA IGTAGDTYYP GSVKGRFTIS RENAKNSLYL QMNSLRAGD TAVYYCARTW FGELYFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYDMHWVRQA TGKGLEWVSA IGTAGDTYYP GSVKGRFTIS RENAKNSLYL QMNSLRAGD TAVYYCARTW FGELYFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVE

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Macromolecule #2: Envelope glycoprotein

MacromoleculeName: Envelope glycoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976 / Strain: Mayinga-76
Molecular weightTheoretical: 35.605875 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: RSIPLGVIHN STLQVSDVDK LVCRDKLSST NQLRSVGLNL EGNGVATDVP SATKRWGFRS GVPPKVVNYE AGEWAENCYN LEIKKPDGS ECLPAAPDGI RGFPRCRYVH KVSGTGPCAG DFAFHKEGAF FLYDRLASTV IYRGTTFAEG VVAFLILPQA K KDFFSSHP ...String:
RSIPLGVIHN STLQVSDVDK LVCRDKLSST NQLRSVGLNL EGNGVATDVP SATKRWGFRS GVPPKVVNYE AGEWAENCYN LEIKKPDGS ECLPAAPDGI RGFPRCRYVH KVSGTGPCAG DFAFHKEGAF FLYDRLASTV IYRGTTFAEG VVAFLILPQA K KDFFSSHP LREPVNATED PSSGYYSTTI RYQATGFGTN ETEYLFEVDN LTYVQLESRF TPQFLLQLNE TIYTSGKRSN TT GKLIWKV NPEIDTTIGE WAFWETKKNL TRKIRSEELS FTVVNTHHQD TGEESASSGK LGLITNTIAG VAGLITGGRR TRR E

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Macromolecule #3: REGN3479 light chain

MacromoleculeName: REGN3479 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.228734 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSIS SFLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQ QSYSTLTFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTEQDSK ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSIS SFLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQ QSYSTLTFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

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Macromolecule #4: REGN3471 light chain

MacromoleculeName: REGN3471 light chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.172803 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLA WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTEFTLT ITSLQAEDV AVYYCQQYYS SPLTFGGGTK VEIKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA L QSGNSQES ...String:
DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLA WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTEFTLT ITSLQAEDV AVYYCQQYYS SPLTFGGGTK VEIKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA L QSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC

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Macromolecule #5: GP2

MacromoleculeName: GP2 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976 / Strain: Mayinga-76
Molecular weightTheoretical: 15.795782 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString:
AIVNAQPKCN PNLHYWTTQD EGAAIGLAWI PYFGPAAEGI YTEGLMHNQD GLICGLRQLA NETTQALQLF LRATTELRTF SILNRKAID FLLQRWGGTC HILGPDCCIE PHDWTKNITD KIDQIIHDFV DKTLPDDDDD K

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Macromolecule #6: REGN3479 heavy chain

MacromoleculeName: REGN3479 heavy chain / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 22.856572 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFTSS SYAMNWVRQA PGKGLEWVST ISGMGGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCAKR GYPHSFDIWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFTSS SYAMNWVRQA PGKGLEWVST ISGMGGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCAKR GYPHSFDIWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVE

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Macromolecule #7: REGN3470 heavy chain

MacromoleculeName: REGN3470 heavy chain / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.187951 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QVQLVESGGG VVQPGRSLRL SCAASGFTFN NYGMHWVRQA PGMGLEWVAV IWHDGSDKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCARN WNLFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS G ALTSGVHT ...String:
QVQLVESGGG VVQPGRSLRL SCAASGFTFN NYGMHWVRQA PGMGLEWVAV IWHDGSDKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAE DTAVYYCARN WNLFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS G ALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVE

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Macromolecule #8: REGN3470 light chain

MacromoleculeName: REGN3470 light chain / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.474076 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: DIQMTQSPSS LSASVGDRIT ITCRASQSIS TYLHWYQQKP GKAPKLLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQ QSFSTPPINF GQGTKLEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIQMTQSPSS LSASVGDRIT ITCRASQSIS TYLHWYQQKP GKAPKLLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQ QSFSTPPINF GQGTKLEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #10: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 10 / Number of copies: 8 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration0.8 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris-base
150.0 mMNaClSodium Chloride
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 3 / Number real images: 6360 / Average exposure time: 6.0 sec. / Average electron dose: 52.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3154
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12) / Number images used: 276715
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5jq3

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7tn9:
Structure of the Inmazeb cocktail and resistance to escape against Ebola virus

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