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- EMDB-27363: nsEM map of hemagglutinin H3 A/Sing/INFIMH/16 complexed with poly... -

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Basic information

Entry
Database: EMDB / ID: EMD-27363
TitlensEM map of hemagglutinin H3 A/Sing/INFIMH/16 complexed with polyclonal Fab samples from individual 182419 at day 28 using Sulfo-SDAD photo-cross-linker
Map datansEM map of hemagglutinin H3 A/Sing/INFIMH/16 complexed with polyclonal Fab samples from individual 182419 at day 28 using Sulfo-SDAD photo-cross-linker
Sample
  • Complex: nsEM map of hemagglutinin H1 A/Mich/045/15 complexed with polyclonal Fab samples from individual 182419 at day 0
Keywordsinfluenza / H1 / nsEM / VIRAL PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsTorrents de la Pena A / Sewall LM / Ward AB
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI136621 United States
Bill & Melinda Gates FoundationINV-004923 United States
Bill & Melinda Gates FoundationINV-002916 United States
CitationJournal: Cell Rep Methods / Year: 2023
Title: Increasing sensitivity of antibody-antigen interactions using photo-cross-linking.
Authors: Alba Torrents de la Peña / Leigh M Sewall / Rebeca de Paiva Froes Rocha / Abigail M Jackson / Payal P Pratap / Sandhya Bangaru / Christopher A Cottrell / Subhasis Mohanty / Albert C Shaw / Andrew B Ward /
Abstract: Understanding antibody-antigen interactions in a polyclonal immune response in humans and animal models is critical for rational vaccine design. Current approaches typically characterize antibodies ...Understanding antibody-antigen interactions in a polyclonal immune response in humans and animal models is critical for rational vaccine design. Current approaches typically characterize antibodies that are functionally relevant or highly abundant. Here, we use photo-cross-linking and single-particle electron microscopy to increase antibody detection and unveil epitopes of low-affinity and low-abundance antibodies, leading to a broader structural characterization of polyclonal immune responses. We employed this approach across three different viral glycoproteins and showed increased sensitivity of detection relative to currently used methods. Results were most noticeable in early and late time points of a polyclonal immune response. Additionally, the use of photo-cross-linking revealed intermediate antibody binding states and demonstrated a distinctive way to study antibody binding mechanisms. This technique can be used to structurally characterize the landscape of a polyclonal immune response of patients in vaccination or post-infection studies at early time points, allowing for rapid iterative design of vaccine immunogens.
History
DepositionJun 19, 2022-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJul 19, 2023-
Current statusJul 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27363.png

Downloads & links

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Map

FileDownload / File: emd_27363.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationnsEM map of hemagglutinin H3 A/Sing/INFIMH/16 complexed with polyclonal Fab samples from individual 182419 at day 28 using Sulfo-SDAD photo-cross-linker
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.06 Å/pix.
x 200 pix.
= 412. Å		2.06 Å/pix.
x 200 pix.
= 412. Å		2.06 Å/pix.
x 200 pix.
= 412. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.018026711 - 0.070879
Average (Standard dev.)0.00012828414 (±0.0025956184)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 412.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: nsEM half map of hemagglutinin H3 A/Sing/INFIMH/16 complexed...

Fileemd_27363_half_map_1.map
AnnotationnsEM half map of hemagglutinin H3 A/Sing/INFIMH/16 complexed with polyclonal Fab samples from individual 182419 at day 28 using Sulfo-SDAD photo-cross-linker
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: nsEM half map of hemagglutinin H3 A/Sing/INFIMH/16 complexed...

Fileemd_27363_half_map_2.map
AnnotationnsEM half map of hemagglutinin H3 A/Sing/INFIMH/16 complexed with polyclonal Fab samples from individual 182419 at day 28 using Sulfo-SDAD photo-cross-linker
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : nsEM map of hemagglutinin H1 A/Mich/045/15 complexed with polyclo...

EntireName: nsEM map of hemagglutinin H1 A/Mich/045/15 complexed with polyclonal Fab samples from individual 182419 at day 0
Components
  • Complex: nsEM map of hemagglutinin H1 A/Mich/045/15 complexed with polyclonal Fab samples from individual 182419 at day 0

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Supramolecule #1: nsEM map of hemagglutinin H1 A/Mich/045/15 complexed with polyclo...

SupramoleculeName: nsEM map of hemagglutinin H1 A/Mich/045/15 complexed with polyclonal Fab samples from individual 182419 at day 0
type: complex / ID: 1 / Parent: 0
Details: HA recombinantly expressed in HEK 293F cells Fab samples isolated from several individuals at different timepoints
Source (natural)Organism: Homo sapiens (human)

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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

Concentration0.02 mg/mL
BufferpH: 7.4 / Component - Concentration: 0.02 0.020 / Component - Formula: TBS / Component - Name: Tris-buffered saline
StainingType: NEGATIVE / Material: Uranyl Formate
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
Details15 ug of H1 A/Mich15/045/15 was complexed overnight with 500 ug of polyclonal Fab samples and SEC purified. The complex peak was concentrated to ~0.02 mg/ml and imaged

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Number grids imaged: 1 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 52000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 95212
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4m4y

Final reconstructionResolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 51619
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: bayesian polishing
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: bayesian polishing

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