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- EMDB-43273: Prefusion-stabilized trimer of FluB-HA under low pH conditions -

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Basic information

Entry
Database: EMDB / ID: EMD-43273
TitlePrefusion-stabilized trimer of FluB-HA under low pH conditions
Map data
Sample
  • Complex: prefusion-stabilized influenza B hemagglutinin
KeywordsCryo-EM / FluB / HA / VIRAL PROTEIN
Biological speciesInfluenza B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsJuraszek J / Milder FJ / Yu X / Blokland S / Overveld D / Tamara S / Bakkers MJG / Rutten L / Sharma S / Langedijk JPM
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: PNAS Nexus / Year: 2024
Title: Engineering a cleaved, prefusion-stabilized influenza B virus hemagglutinin by identification and locking of all six pH switches.
Authors: Jarek Juraszek / Fin J Milder / Xiaodi Yu / Sven Blokland / Daan van Overveld / Pravien Abeywickrema / Sem Tamara / Sujata Sharma / Lucy Rutten / Mark J G Bakkers / Johannes P M Langedijk /
Abstract: Vaccine components based on viral fusion proteins require high stability of the native prefusion conformation for optimal potency and manufacturability. In the case of influenza B virus hemagglutinin ...Vaccine components based on viral fusion proteins require high stability of the native prefusion conformation for optimal potency and manufacturability. In the case of influenza B virus hemagglutinin (HA), the stem's conformation relies on efficient cleavage. In this study, we identified six pH-sensitive regions distributed across the entire ectodomain where protonated histidines assume either a repulsive or an attractive role. Substitutions in these areas enhanced the protein's expression, quality, and stability in its prefusion trimeric state. Importantly, this stabilization enabled the production of a cleavable HA0, which is further processed into HA1 and HA2 by furin during exocytic pathway passage, thereby facilitating correct folding, increased stability, and screening for additional stabilizing substitutions in the core of the metastable fusion domain. Cryo-EM analysis at neutral and low pH revealed a previously unnoticed pH switch involving the C-terminal residues of the natively cleaved HA1. This switch keeps the fusion peptide in a clamped state at neutral pH, averting premature conformational shift. Our findings shed light on new strategies for possible improvements of recombinant or genetic-based influenza B vaccines.
History
DepositionJan 5, 2024-
Header (metadata) releaseDec 25, 2024-
Map releaseDec 25, 2024-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43273.png

Downloads & links

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Map

FileDownload / File: emd_43273.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 280 pix.
= 254.8 Å		0.91 Å/pix.
x 280 pix.
= 254.8 Å		0.91 Å/pix.
x 280 pix.
= 254.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-2.9658747 - 3.6047554
Average (Standard dev.)-0.0004357223 (±0.083004415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 254.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43273_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_43273_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : prefusion-stabilized influenza B hemagglutinin

EntireName: prefusion-stabilized influenza B hemagglutinin
Components
  • Complex: prefusion-stabilized influenza B hemagglutinin

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Supramolecule #1: prefusion-stabilized influenza B hemagglutinin

SupramoleculeName: prefusion-stabilized influenza B hemagglutinin / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Influenza B virus

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1185383
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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