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- EMDB-46833: Negative stain map of cH5/1 apo -

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Basic information

Entry
Database: EMDB / ID: EMD-46833
TitleNegative stain map of cH5/1 apo
Map dataNegative stain map of global refinement of particle stack used to sort cH5/1 apo
Sample
  • Virus: Influenza A virus (A/California/04/2009(H1N1))
Keywordsinfluenza / hemmagglutinin / HA / Fab complex / viral fusion protein / chimeric hemmagglutinin / VIRAL Protein
Biological speciesInfluenza A virus (A/California/04/2009(H1N1))
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsHan J / Rodriguez AJ / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)75N93109C00051 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural characterization of influenza group 1 chimeric hemagglutinins as broad vaccine immunogens.
Authors: Yen Thi Kim Nguyen / Xueyong Zhu / Julianna Han / Alesandra J Rodriguez / Weina Sun / Wenli Yu / Peter Palese / Florian Krammer / Andrew B Ward / Ian A Wilson /
Abstract: Chimeric hemagglutinins (cHA) appear to be promising for the design and development of universal influenza vaccines. Influenza A group 1 cHAs, cH5/1, cH8/1, and cH11/1, comprising an H1 stem attached ...Chimeric hemagglutinins (cHA) appear to be promising for the design and development of universal influenza vaccines. Influenza A group 1 cHAs, cH5/1, cH8/1, and cH11/1, comprising an H1 stem attached to either an H5, H8, or H11 globular head, have been used sequentially as vaccine immunogens in human clinical trials and induced high levels of broadly protective antibodies. Using X-ray crystallography and negative-stain electron microscopy, we determined structures of cH5/1, cH8/1, and cH11/1 HAs in their apo (unliganded) and antibody Fab-bound states. Stem-reactive antibodies 3E1 and 31.b.09 recognize their cognate epitopes in cH5/1, cH8/1, and cH11/1 HAs. However, with cH5/1, the head domains are rotated by 35 to 45° around the threefold axis of the HA trimer compared to native HA with a more splayed-open conformation at the stem base. cH11/1 with 3E1 is structurally more native-like but resembles cH5/1 with 31.b.09, whereas cH8/1 with 31.b.09 exhibited a range of closed-to-open stem configurations with some separation of head and stem domains. Furthermore, all of these group 1 cHAs effectively bound a broad head trimer interface antibody and other broad stem antibodies. Thus, the cHAs exhibit structural plasticity without compromising the stem and head trimer interface epitopes for elicitation of influenza A group 1 cross-reactive antibodies.
History
DepositionSep 2, 2024-
Header (metadata) releaseFeb 26, 2025-
Map releaseFeb 26, 2025-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46833.png

Downloads & links

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Map

FileDownload / File: emd_46833.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain map of global refinement of particle stack used to sort cH5/1 apo
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.77 Å/pix.
x 180 pix.
= 318.6 Å		1.77 Å/pix.
x 180 pix.
= 318.6 Å		1.77 Å/pix.
x 180 pix.
= 318.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0132
Minimum - Maximum-0.033600938 - 0.076077595
Average (Standard dev.)0.00011285506 (±0.0038012539)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 318.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Negative stain map of cH5/1 closed apo

Fileemd_46833_additional_1.map
AnnotationNegative stain map of cH5/1 closed apo
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Negative stain map of cH5/1 opening apo

Fileemd_46833_additional_2.map
AnnotationNegative stain map of cH5/1 opening apo
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Negative stain half map A of global refinement...

Fileemd_46833_half_map_1.map
AnnotationNegative stain half map A of global refinement of particle stack used to sort cH5/1 apo
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Negative stain half map B of global refinement...

Fileemd_46833_half_map_2.map
AnnotationNegative stain half map B of global refinement of particle stack used to sort cH5/1 apo
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Influenza A virus (A/California/04/2009(H1N1))

EntireName: Influenza A virus (A/California/04/2009(H1N1))
Components
  • Virus: Influenza A virus (A/California/04/2009(H1N1))

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Supramolecule #1: Influenza A virus (A/California/04/2009(H1N1))

SupramoleculeName: Influenza A virus (A/California/04/2009(H1N1)) / type: virus / ID: 1 / Parent: 0
Details: Recombinantly expressed protein cH5/1: HA head domain of A/Vietnam/1203/2004(H5N1) and HA stem domain of A/California/04/2009(H1N1)
NCBI-ID: 641501
Sci species name: Influenza A virus (A/California/04/2009(H1N1))
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No

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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: 2% w/v uranyl formate
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.2 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

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

4m4y

Final reconstructionResolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 32600
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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