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- EMDB-15073: Rabies virus glycoprotein in complex with Fab fragments of 17C7 a... -

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Basic information

Entry
Database: EMDB / ID: EMD-15073
TitleRabies virus glycoprotein in complex with Fab fragments of 17C7 and 1112-1 neutralizing antibodies
Map dataSharpened final cryo-EM map
Sample
  • Complex: Rabies virus glycoprotein (chain A) in complex with Fab 1112-1 (chains B and C) and Fab 17C7 (chains D and E)
    • Protein or peptide: Glycoprotein
    • Protein or peptide: Fab 17C7 heavy chain variable domain
    • Protein or peptide: Fab 17C7 light chain variable domain
    • Protein or peptide: Fab 1112-1 light chain variable domain
    • Protein or peptide: Fab 1112-1 heavy chain variable domain
Function / homologyRhabdovirus glycoprotein / Rhabdovirus spike glycoprotein / membrane => GO:0016020 / viral envelope / virion membrane / Glycoprotein
Function and homology information
Biological speciesRabies lyssavirus / Rabies virus strain Pasteur vaccin / Homo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsNg WM / Fedosyuk S / English S / Augusto G / Berg A / Thorley L / Haselon AS / Segireddy RR / Bowden TA / Douglas AD
Funding support United Kingdom, European Union, 7 items
OrganizationGrant numberCountry
Wellcome Trust220679/Z/20/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/P017339/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/S007555/1 United Kingdom
Marie Sklodowska-Curie Actions, FragNET ITN840866European Union
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
Wellcome Trust203141/Z/16/Z United Kingdom
CitationJournal: Cell Host Microbe / Year: 2022
Title: Structure of trimeric pre-fusion rabies virus glycoprotein in complex with two protective antibodies.
Authors: Weng M Ng / Sofiya Fedosyuk / Solomon English / Gilles Augusto / Adam Berg / Luke Thorley / Anna-Sophie Haselon / Rameswara R Segireddy / Thomas A Bowden / Alexander D Douglas /
Abstract: Rabies virus (RABV) causes lethal encephalitis and is responsible for approximately 60,000 deaths per year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates host- ...Rabies virus (RABV) causes lethal encephalitis and is responsible for approximately 60,000 deaths per year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates host-cell entry. RABV-G's pre-fusion trimeric conformation displays epitopes bound by protective neutralizing antibodies that can be induced by vaccination or passively administered for post-exposure prophylaxis. We report a 2.8-Å structure of a RABV-G trimer in the pre-fusion conformation, in complex with two neutralizing and protective monoclonal antibodies, 17C7 and 1112-1, that recognize distinct epitopes. One of these antibodies is a licensed prophylactic (17C7, Rabishield), which we show locks the protein in pre-fusion conformation. Targeted mutations can similarly stabilize RABV-G in the pre-fusion conformation, a key step toward structure-guided vaccine design. These data reveal the higher-order architecture of a key therapeutic target and the structural basis of neutralization by antibodies binding two key antigenic sites, and this will facilitate the development of improved vaccines and prophylactic antibodies.
History
DepositionJun 1, 2022-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateSep 28, 2022-
Current statusSep 28, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15073.png

Downloads & links

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Map

FileDownload / File: emd_15073.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened final cryo-EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 400 pix.
= 424. Å		1.06 Å/pix.
x 400 pix.
= 424. Å		1.06 Å/pix.
x 400 pix.
= 424. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-5.263432 - 7.1617103
Average (Standard dev.)-5.0908813e-05 (±0.0987536)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 423.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15073_msk_1.map
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Mask #2

Fileemd_15073_msk_2.map
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Half map: Half map A

Fileemd_15073_half_map_1.map
AnnotationHalf map A
Projections & Slices
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Half map: Half map B

Fileemd_15073_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : Rabies virus glycoprotein (chain A) in complex with Fab 1112-1 (c...

EntireName: Rabies virus glycoprotein (chain A) in complex with Fab 1112-1 (chains B and C) and Fab 17C7 (chains D and E)
Components
  • Complex: Rabies virus glycoprotein (chain A) in complex with Fab 1112-1 (chains B and C) and Fab 17C7 (chains D and E)
    • Protein or peptide: Glycoprotein
    • Protein or peptide: Fab 17C7 heavy chain variable domain
    • Protein or peptide: Fab 17C7 light chain variable domain
    • Protein or peptide: Fab 1112-1 light chain variable domain
    • Protein or peptide: Fab 1112-1 heavy chain variable domain

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Supramolecule #1: Rabies virus glycoprotein (chain A) in complex with Fab 1112-1 (c...

SupramoleculeName: Rabies virus glycoprotein (chain A) in complex with Fab 1112-1 (chains B and C) and Fab 17C7 (chains D and E)
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rabies lyssavirus
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 330 KDa

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Macromolecule #1: Glycoprotein

MacromoleculeName: Glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rabies virus strain Pasteur vaccin / Strain: Pasteur vaccins / PV
Molecular weightTheoretical: 56.494551 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: KFPIYTIPDK LGPWSPIDIH HLSCPNNLVV EDEGCTNLSG FSYMELKVGY ISAIKMNGFT CTGVVTEAET YTNFVGYVTT TFKRKHFRP TPDACRAAYN WKMAGDPRYE ESLHNPYPDY HWLRTVKTTK ESLVIISPSV ADLDPYDRSL HSPVFPGGNC S GVAVSSTY ...String:
KFPIYTIPDK LGPWSPIDIH HLSCPNNLVV EDEGCTNLSG FSYMELKVGY ISAIKMNGFT CTGVVTEAET YTNFVGYVTT TFKRKHFRP TPDACRAAYN WKMAGDPRYE ESLHNPYPDY HWLRTVKTTK ESLVIISPSV ADLDPYDRSL HSPVFPGGNC S GVAVSSTY CSTNHDYTIW MPENPRLGMS CDIFTNSRGK RASKGSETCG FVDERGLYKS LKGACKLKLC GVLGLRLMDG TW VAMQTSN ETKWCPPGQL VNLHDFRSDE IEPLVVEELV KKREECLDAL ESIMTTKSVS FRRLSHLRKL VPGFGKAYTI FNK TLMEAD AHYKSVRTWN EIIPSKGCLR VGGRCHPHVN GVFFNGIILG PDGNVLIPEM QSSLLQQHME LLVSSVIPLM HPLA DPSTV FKNGDEAEDF VEVHLPDVHE RISGVDLGLP NWGKYVLLSA GALTALMLII FLMTCWRRVN RSEPTQHNLR GTGRE VSVT PQSGKIISSW ESHKSGGETR L

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Macromolecule #2: Fab 17C7 heavy chain variable domain

MacromoleculeName: Fab 17C7 heavy chain variable domain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.227727 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLVESGGG VVQPGRSLRL SCAASGFTFS TYAMHWVRQA PGKGLEWVAV VSYDGRTKDY ADSVKGRFTI SRDNSKNTLY LQMNSLRTE DTAVYFCARE RFSGAYFDYW GQGTLVTVSS

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Macromolecule #3: Fab 17C7 light chain variable domain

MacromoleculeName: Fab 17C7 light chain variable domain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.754044 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYSCQ QRNNWPPTFG GGTKVEIKRA

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Macromolecule #4: Fab 1112-1 light chain variable domain

MacromoleculeName: Fab 1112-1 light chain variable domain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.668073 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
DIVMTQSHKF MSTSIGDRVS ITCKASQDVS TAVAWYRQKP GQSPKLLIYS ASYRSTGVPD RFTGSGSGTD FTFTISSVQA EDLAVFYCQ QHYSAPLTFG AGTKLELK

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Macromolecule #5: Fab 1112-1 heavy chain variable domain

MacromoleculeName: Fab 1112-1 heavy chain variable domain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.426977 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
QVQLQQPGAE LLKPGTSMKL SCKASGYTFS NYWMHWVKLR PGQGFEWIGE INPFNGGTNF NEKFKSKATL TVDRSSSTAY MQLSSVTSE DSAVYYCTIP LSDYGDWFFA VWGAGTTVTV SS

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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

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
50.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Blot for 3.5 seconds at -15 N blot force before plunging..
DetailsPurified sample was concentrated and mixed with 0.07% n-octyl-beta-d-glucoside to final concentrations of 1 mg/mL.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 12884 / Average exposure time: 3.7 sec. / Average electron dose: 44.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3112037
Details: Particles were automatically picked using circular blobs with a diameter of 80-150 Angstrom on cryoSPARC.
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: INSILICO MODEL
Details: Initial models were generated using cryoSPARC ab-initio reconstruction.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) / Details: cryoSPARC heterogeneous refinement was used.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) / Details: cryoSPARC non-uniform refinement was used.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Details: cryoSPARC non-uniform refinement was used. / Number images used: 458014
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel building was performed with Coot and refined with Phenix.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 108
Output model

PDB-8a1e:
Rabies virus glycoprotein in complex with Fab fragments of 17C7 and 1112-1 neutralizing antibodies

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