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- EMDB-40844: Negative stain EM map 1 of polyclonal serum from rabbit immunized... -

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Entry
Database: EMDB / ID: EMD-40844
TitleNegative stain EM map 1 of polyclonal serum from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon
Map dataNegative stain EM map of polyclonal serum from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon.
Sample
  • Complex: polyclonal serum cleaved to Fabs from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon
    • Complex: polyclonal serum cleaved to Fabs from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5
    • Complex: Malaysia54 HA-foldon
KeywordsFab / Serum / Influenza Hemagglutinin / Immune Complex / IMMUNE SYSTEM
Biological speciesOryctolagus cuniculus (rabbit) / Influenza A virus
Methodsingle particle reconstruction / negative staining / Resolution: 15.4 Å
AuthorsDosey A / King NP
Funding support United States, 1 items
OrganizationGrant numberCountry
Open Philanthropy68-0683 United States
CitationJournal: Cell Rep / Year: 2023
Title: Combinatorial immune refocusing within the influenza hemagglutinin RBD improves cross-neutralizing antibody responses.
Authors: Annie Dosey / Daniel Ellis / Seyhan Boyoglu-Barnum / Hubza Syeda / Mason Saunders / Michael J Watson / John C Kraft / Minh N Pham / Miklos Guttman / Kelly K Lee / Masaru Kanekiyo / Neil P King /
Abstract: The receptor-binding domain (RBD) of influenza virus hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies. Here, we evaluate the ability of several immunofocusing ...The receptor-binding domain (RBD) of influenza virus hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies. Here, we evaluate the ability of several immunofocusing techniques to enhance the functional breadth of vaccine-elicited immune responses against the HA RBD. We present a series of "trihead" nanoparticle immunogens that display native-like closed trimeric RBDs from the HAs of several H1N1 influenza viruses. The series includes hyperglycosylated and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the receptor-binding site periphery. Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicit higher hemagglutination inhibition (HAI) and neutralizing activity than the corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation. By contrast, mosaic nanoparticle display and antigen hypervariation do not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Our results yield important insights into antibody responses against the RBD and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.
History
DepositionMay 22, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40844.png

Downloads & links

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Map

FileDownload / File: emd_40844.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM map of polyclonal serum from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.49 Å/pix.
x 192 pix.
= 478.08 Å		2.49 Å/pix.
x 192 pix.
= 478.08 Å		2.49 Å/pix.
x 192 pix.
= 478.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.49 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.15
Minimum - Maximum-0.95652115 - 3.3231928
Average (Standard dev.)0.000509776 (±0.15832403)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 478.08002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Negative stain EM map of polyclonal serum from...

Fileemd_40844_half_map_1.map
AnnotationNegative stain EM map of polyclonal serum from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Negative stain EM map of polyclonal serum from...

Fileemd_40844_half_map_2.map
AnnotationNegative stain EM map of polyclonal serum from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : polyclonal serum cleaved to Fabs from rabbit immunized with Trihe...

EntireName: polyclonal serum cleaved to Fabs from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon
Components
  • Complex: polyclonal serum cleaved to Fabs from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon
    • Complex: polyclonal serum cleaved to Fabs from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5
    • Complex: Malaysia54 HA-foldon

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Supramolecule #1: polyclonal serum cleaved to Fabs from rabbit immunized with Trihe...

SupramoleculeName: polyclonal serum cleaved to Fabs from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5 in complex with Malaysia54 HA-foldon
type: complex / ID: 1 / Parent: 0

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Supramolecule #2: polyclonal serum cleaved to Fabs from rabbit immunized with Trihe...

SupramoleculeName: polyclonal serum cleaved to Fabs from rabbit immunized with Trihead-Hypervariable-Mosaic-I53_dn5
type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #3: Malaysia54 HA-foldon

SupramoleculeName: Malaysia54 HA-foldon / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Influenza A virus / Strain: Malaysia54

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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

BufferpH: 8
StainingType: NEGATIVE / Material: Uranyl Acetate

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Electron microscopy

MicroscopeTFS TALOS L120C
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Talos L120C / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab Initio model generation
Final reconstructionResolution.type: BY AUTHOR / Resolution: 15.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 641
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC

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