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- EMDB-46788: VFLIP Spike Trimer with 4C12-B12 -

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Basic information

Entry
Database: EMDB / ID: EMD-46788
TitleVFLIP Spike Trimer with 4C12-B12
Map data
Sample
  • Complex: VFLIP Spike trimer with 4C12-B12 FAB
KeywordsCryoEM / Fab / Antiviral / SARs-Cov2 / VIRAL PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsSobti M / Stewart AG
Funding support Australia, 1 items
OrganizationGrant numberCountry
Australian Research Council (ARC) Australia
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Affinity maturation endows potent activity onto class 6 SARS-CoV-2 broadly neutralizing antibodies.
Authors: Ohan Mazigi / David B Langley / Jake Y Henry / Deborah L Burnett / Meghna Sobti / Gregory J Walker / Romain Rouet / Harikrishnan Balachandran / Helen Lenthall / Jennifer Jackson / Stephanie ...Authors: Ohan Mazigi / David B Langley / Jake Y Henry / Deborah L Burnett / Meghna Sobti / Gregory J Walker / Romain Rouet / Harikrishnan Balachandran / Helen Lenthall / Jennifer Jackson / Stephanie Ubiparipovic / Peter Schofield / Simon H J Brown / Sebastian R Schulz / Markus Hoffmann / Stefan Pöhlmann / Jeffrey Post / Marianne Martinello / Golo Ahlenstiel / Anthony Kelleher / William D Rawlinson / Stuart G Turville / Rowena A Bull / Alastair G Stewart / Hans-Martin Jäck / Christopher C Goodnow / Daniel Christ /
Abstract: The emergence of SARS-CoV-2 variants of concern (VOCs) has greatly diminished the neutralizing activity of previously FDA-approved monoclonal antibodies (mAbs), including that of antibody cocktails ...The emergence of SARS-CoV-2 variants of concern (VOCs) has greatly diminished the neutralizing activity of previously FDA-approved monoclonal antibodies (mAbs), including that of antibody cocktails and of first-generation broadly neutralizing antibodies such as S309 (Sotrovimab). In contrast, antibodies targeting cryptic conformational epitopes of the receptor binding domain (RBD) have demonstrated broad activity against emerging variants, but exert only moderate neutralizing activity, which has so far hindered clinical development. Here, we utilize in vitro display technology to identify and affinity-mature antibodies targeting the cryptic class 6 epitope, accessible only in the "up" conformation of the SARS-CoV-2 spike trimer. Increasing antibody affinity into the low picomolar range endowed potent neutralization of VOCs and protection of hACE2 mice from viral challenge. Cryoelectron microscopy and crystal structures of two affinity-matured antibodies (4C12-B12 and 4G1-C2) in complex with RBD highlighted binding modes and epitopes distal from mutational hotspots commonly overserved in VOCs, providing direct structural insights into the observed mutational resistance. Moreover, we further demonstrate that antibodies targeting the class 6 epitope, rather than being an artifact of in vitro selection, are common in the IgG1 memory B cell repertoire of convalescent patients and can be induced in human antibody V-gene transgenic mice through immunization. Our results highlight the importance of very high (picomolar) affinity in the development of neutralizing antibodies and vaccines and suggest an affinity threshold in the provision of broad and long-lasting immunity against SARS-CoV-2.
History
DepositionAug 28, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46788.png

Downloads & links

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Map

FileDownload / File: emd_46788.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.2674856 - 1.6126351
Average (Standard dev.)0.0000131843135 (±0.03493638)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_46788_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_46788_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : VFLIP Spike trimer with 4C12-B12 FAB

EntireName: VFLIP Spike trimer with 4C12-B12 FAB
Components
  • Complex: VFLIP Spike trimer with 4C12-B12 FAB

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Supramolecule #1: VFLIP Spike trimer with 4C12-B12 FAB

SupramoleculeName: VFLIP Spike trimer with 4C12-B12 FAB / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 72509
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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