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- EMDB-7889: Negative Stain EM map of polyclonal serum in complex with BG505 S... -

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Basic information

Entry
Database: EMDB / ID: EMD-7889
TitleNegative Stain EM map of polyclonal serum in complex with BG505 SOSIP.664 from rabbit 3419 at post boost 2.
Map dataNegative stain EM map of polyclonal serum in complex with BG505 SOSIP.664 from rabbit 3419 at post boost 2
Sample
  • Complex: Negative stain EM map of polyclonal serum in complex with BG505 SOSIP.664 from rabbit 3419 at post boost 2. Serum digested and Fab purified before adding trimer.
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsWard AB / Turner HL
CitationJournal: Immunity / Year: 2018
Title: Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization.
Authors: Matteo Bianchi / Hannah L Turner / Bartek Nogal / Christopher A Cottrell / David Oyen / Matthias Pauthner / Raiza Bastidas / Rebecca Nedellec / Laura E McCoy / Ian A Wilson / Dennis R Burton ...Authors: Matteo Bianchi / Hannah L Turner / Bartek Nogal / Christopher A Cottrell / David Oyen / Matthias Pauthner / Raiza Bastidas / Rebecca Nedellec / Laura E McCoy / Ian A Wilson / Dennis R Burton / Andrew B Ward / Lars Hangartner /
Abstract: Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a ...Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.
History
DepositionMay 23, 2018-
Header (metadata) releaseJun 20, 2018-
Map releaseSep 5, 2018-
UpdateSep 5, 2018-
Current statusSep 5, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0253
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0253
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7889.png

Downloads & links

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Map

FileDownload / File: emd_7889.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM map of polyclonal serum in complex with BG505 SOSIP.664 from rabbit 3419 at post boost 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.05 Å/pix.
x 144 pix.
= 295.2 Å		2.05 Å/pix.
x 144 pix.
= 295.2 Å		2.05 Å/pix.
x 144 pix.
= 295.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0253 / Movie #1: 0.0253
Minimum - Maximum-0.032704506 - 0.07558903
Average (Standard dev.)0.000573239 (±0.0071558002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 295.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z295.200295.200295.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.0330.0760.001

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Supplemental data

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Sample components

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Entire : Negative stain EM map of polyclonal serum in complex with BG505 S...

EntireName: Negative stain EM map of polyclonal serum in complex with BG505 SOSIP.664 from rabbit 3419 at post boost 2. Serum digested and Fab purified before adding trimer.
Components
  • Complex: Negative stain EM map of polyclonal serum in complex with BG505 SOSIP.664 from rabbit 3419 at post boost 2. Serum digested and Fab purified before adding trimer.

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Supramolecule #1: Negative stain EM map of polyclonal serum in complex with BG505 S...

SupramoleculeName: Negative stain EM map of polyclonal serum in complex with BG505 SOSIP.664 from rabbit 3419 at post boost 2. Serum digested and Fab purified before adding trimer.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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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

Concentration0.063 mg/mL
BufferpH: 7.4 / Component - Formula: TBS
StainingType: NEGATIVE / Material: Uranyl formate
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CELLULOSE ACETATE / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 50 / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 17487
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final 3D classificationSoftware - Name: RELION (ver. 2.1)

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