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- EMDB-43174: GPCysRRLL-I53-50A in complex with rabbit 187 wk 30 GPC-A, base, a... -

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Basic information

Entry
Database: EMDB / ID: EMD-43174
TitleGPCysRRLL-I53-50A in complex with rabbit 187 wk 30 GPC-A, base, and fusion peptide epitope pAbs
Map datarabbit 187 wk30 GPC-A pAb
Sample
  • Complex: GPCysRRLL-I53-50A+8.9F Fab in complex with rabbit 187 wk30 GPC-A, base, and fusion peptide pAbs
KeywordsLassa virus / polyclonal antibody / glycoprotein / VIRAL PROTEIN
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsBrouwer PJM / Perrett HR / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI171438 United States
CitationJournal: Cell Rep / Year: 2024
Title: Defining bottlenecks and opportunities for Lassa virus neutralization by structural profiling of vaccine-induced polyclonal antibody responses.
Authors: Philip J M Brouwer / Hailee R Perrett / Tim Beaumont / Haye Nijhuis / Sabine Kruijer / Judith A Burger / Ilja Bontjer / Wen-Hsin Lee / James A Ferguson / Martin Schauflinger / Helena Müller- ...Authors: Philip J M Brouwer / Hailee R Perrett / Tim Beaumont / Haye Nijhuis / Sabine Kruijer / Judith A Burger / Ilja Bontjer / Wen-Hsin Lee / James A Ferguson / Martin Schauflinger / Helena Müller-Kräuter / Rogier W Sanders / Thomas Strecker / Marit J van Gils / Andrew B Ward /
Abstract: Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus ...Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccine candidates have generally been unsuccessful at doing so, and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron microscopy-based epitope mapping workflow that enables high-resolution structural characterization of polyclonal antibodies to the GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization that involve epitopes of the GPC-A competition cluster. Furthermore, by identifying undescribed immunogenic off-target epitopes, we expose the challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.
History
DepositionDec 20, 2023-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43174.png

Downloads & links

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Map

FileDownload / File: emd_43174.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationrabbit 187 wk30 GPC-A pAb
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.06 Å/pix.
x 150 pix.
= 309. Å		2.06 Å/pix.
x 150 pix.
= 309. Å		2.06 Å/pix.
x 150 pix.
= 309. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0283
Minimum - Maximum-0.024859611 - 0.097398855
Average (Standard dev.)0.0004901422 (±0.0057656216)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 309.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: rabbit 187 wk30 base pAb

Fileemd_43174_additional_1.map
Annotationrabbit 187 wk30 base pAb
Projections & Slices
AxesZYX

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Additional map: rabbit 187 wk30 fusion peptide pAb

Fileemd_43174_additional_2.map
Annotationrabbit 187 wk30 fusion peptide pAb
Projections & Slices
AxesZYX

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Half map: rabbit 187 wk30 GPC-A pAb

Fileemd_43174_half_map_1.map
Annotationrabbit 187 wk30 GPC-A pAb
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: rabbit 187 wk30 GPC-A pAb

Fileemd_43174_half_map_2.map
Annotationrabbit 187 wk30 GPC-A pAb
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GPCysRRLL-I53-50A+8.9F Fab in complex with rabbit 187 wk30 GPC-A,...

EntireName: GPCysRRLL-I53-50A+8.9F Fab in complex with rabbit 187 wk30 GPC-A, base, and fusion peptide pAbs
Components
  • Complex: GPCysRRLL-I53-50A+8.9F Fab in complex with rabbit 187 wk30 GPC-A, base, and fusion peptide pAbs

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Supramolecule #1: GPCysRRLL-I53-50A+8.9F Fab in complex with rabbit 187 wk30 GPC-A,...

SupramoleculeName: GPCysRRLL-I53-50A+8.9F Fab in complex with rabbit 187 wk30 GPC-A, base, and fusion peptide pAbs
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.015 mg/mL
BufferpH: 7.4
StainingType: NEGATIVE / Material: Uranyl Formate / Details: 2% w/v
GridModel: Homemade / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8er3
PDB Unreleased entry


Details: The startup model also contains the I53-50A scaffold (7SGE) and a constant domain for 8.9F Fab was added by overlaying with full rabbit Fab (4ZTP). The overall complex was low pass filtered in UCSF Chimera.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION / Number images used: 16689
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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