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- EMDB-42046: H7N9 A/Shanghai/2/2013 complexed with mouse polyclonal Fab - wk 1... -

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Basic information

Entry
Database: EMDB / ID: EMD-42046
TitleH7N9 A/Shanghai/2/2013 complexed with mouse polyclonal Fab - wk 12, control immunogen
Map data
Sample
  • Complex: H7HA A/Shanghai/2/2013 immune complex with mouse polyclonal Fab - week 12, control immunogen group
    • Complex: H7N9 A/Shanghai/2/2013
    • Complex: Polyclonal Fab
    • Protein or peptide: H7N9 A/Shanghai/2/2013
KeywordsComplex / Fab / Hemagglutinin / polyclonal / IMMUNE SYSTEM
Biological speciesH7N9 subtype (virus) / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 23.5 Å
AuthorsCarter JJ / Barnes CO
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)GT15335 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Vaccine design via antigen reorientation.
Authors: Duo Xu / Joshua J Carter / Chunfeng Li / Ashley Utz / Payton A B Weidenbacher / Shaogeng Tang / Mrinmoy Sanyal / Bali Pulendran / Christopher O Barnes / Peter S Kim /
Abstract: A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily ...A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.
History
DepositionSep 20, 2023-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42046.png

Downloads & links

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Map

FileDownload / File: emd_42046.map.gz / Format: CCP4 / Size: 844.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
7.05 Å/pix.
x 60 pix.
= 423. Å		7.05 Å/pix.
x 60 pix.
= 423. Å		7.05 Å/pix.
x 60 pix.
= 423. Å

Surface

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Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.533
Minimum - Maximum-1.053987 - 3.6205742
Average (Standard dev.)0.0027328825 (±0.1079071)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 423.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_42046_half_map_1.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: #2

Fileemd_42046_half_map_2.map
Projections & Slices
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Sample components

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Entire : H7HA A/Shanghai/2/2013 immune complex with mouse polyclonal Fab -...

EntireName: H7HA A/Shanghai/2/2013 immune complex with mouse polyclonal Fab - week 12, control immunogen group
Components
  • Complex: H7HA A/Shanghai/2/2013 immune complex with mouse polyclonal Fab - week 12, control immunogen group
    • Complex: H7N9 A/Shanghai/2/2013
    • Complex: Polyclonal Fab
    • Protein or peptide: H7N9 A/Shanghai/2/2013

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Supramolecule #1: H7HA A/Shanghai/2/2013 immune complex with mouse polyclonal Fab -...

SupramoleculeName: H7HA A/Shanghai/2/2013 immune complex with mouse polyclonal Fab - week 12, control immunogen group
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Recombinantly expressed H7HA with polyclonal Fabs generated from pooled H2HA-immunized mouse antisera

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Supramolecule #2: H7N9 A/Shanghai/2/2013

SupramoleculeName: H7N9 A/Shanghai/2/2013 / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: H7N9 subtype (virus)

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Supramolecule #3: Polyclonal Fab

SupramoleculeName: Polyclonal Fab / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: H7N9 A/Shanghai/2/2013

MacromoleculeName: H7N9 A/Shanghai/2/2013 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: H7N9 subtype (virus)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNTQILVFAL IAIIPTNADK ICLGHHAVSN GTKVNTLTER GVEVVNATET VERTNIPRIC SKGKRTVDLG QCGLLGTITG PPQCDQFLEF SADLIIERRE GSDVCFPGKF VNEEALRQIL RESGGIDKEA MGFTYSGIRT NGATSACRRS GSSFYAEMKW LLSNTDNAAF ...String:
MNTQILVFAL IAIIPTNADK ICLGHHAVSN GTKVNTLTER GVEVVNATET VERTNIPRIC SKGKRTVDLG QCGLLGTITG PPQCDQFLEF SADLIIERRE GSDVCFPGKF VNEEALRQIL RESGGIDKEA MGFTYSGIRT NGATSACRRS GSSFYAEMKW LLSNTDNAAF PQMTKSYKNT RKSPALIVWG IHHSVSTAEQ TKLYGSGNKL VTVGSSNYQQ SFVPSPGARP QVNGLSGRID FHWLMLNPND TVTFSFNGAF IAPDRASFLR GKSMGIQSGV QVDANCEGDC YHSGGTIISN LPFQNIDSRA VGKCPRYVKQ RSLLLATGMK NVPEIPKGRG LFGAIAGFIE NGWEGLIDGW YGFRHQNAQG EGTAADYKST QSAIDQITGK LNRLIEKTNQ QFELIDNEFN EVEKQIGNVI NWTRDSITEV WSYNAELLVA MENQHTIDLA DSEMDKLYER VKRQLRENAE EDGTGCFEIF HKCDDDCMAS IRNNTYDHSK YREEAMQNRI QIDGSMKQIE DKIEEILSKI YHIENEIARI KKLIGEVASS SGLNDIFEAQ KIEWHEAHHH HHHG

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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
StainingType: NEGATIVE / Material: Uranyl Formate

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 15.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Stochastic gradient descent ab initio in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 23.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 7817
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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