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- EMDB-7903: Negative stain EM map of BG505 SOSIP.644 in complex with PGT121 a... -

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Basic information

Entry
Database: EMDB / ID: EMD-7903
TitleNegative stain EM map of BG505 SOSIP.644 in complex with PGT121 and 12N Fabs
Map dataNegative stain EM map of BG505 SOSIP.644 in complex with PGT121 and 12N Fabs
Sample
  • Complex: Complex of BG505 SOSIP.664 with PGT121 and 12N fabs
Biological speciesHuman immunodeficiency virus
Methodsingle particle reconstruction / negative staining / Resolution: 17.36 Å
AuthorsTurner HL / Ward AB / Nogal B / Cottrell C
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI100663 United States
Bill & Melinda Gates FoundationOPP1115782 United States
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-
UpdateFeb 3, 2021-
Current statusFeb 3, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0243
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0243
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7903.png

Downloads & links

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Map

FileDownload / File: emd_7903.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM map of BG505 SOSIP.644 in complex with PGT121 and 12N Fabs
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 EMDB: 0.0243 / Movie #1: 0.0243
Minimum - Maximum-0.038766615 - 0.086525105
Average (Standard dev.)0.0008592084 (±0.008452594)
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
start NX/NY/NZ-210-210-210
NX/NY/NZ420420420
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.0390.0870.001

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Supplemental data

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Sample components

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Entire : Complex of BG505 SOSIP.664 with PGT121 and 12N fabs

EntireName: Complex of BG505 SOSIP.664 with PGT121 and 12N fabs
Components
  • Complex: Complex of BG505 SOSIP.664 with PGT121 and 12N fabs

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Supramolecule #1: Complex of BG505 SOSIP.664 with PGT121 and 12N fabs

SupramoleculeName: Complex of BG505 SOSIP.664 with PGT121 and 12N fabs / type: complex / ID: 1 / Parent: 0
Details: Serum was purified through SEC after adding excess PGT121 and 12N fabs
Source (natural)Organism: Human immunodeficiency virus
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F

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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

Concentration0.077 mg/mL
BufferpH: 7.4 / Details: TBS
StainingType: NEGATIVE / Material: Uranyl Formate / Details: at 2%
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CELLULOSE ACETATE / Pretreatment - Type: GLOW DISCHARGE
DetailsSample in TBS, stained with UF

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-46 / Number grids imaged: 1 / Number real images: 222 / Average exposure time: 0.5 sec. / 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

Particle selectionDetails: DogPicker was used to pick particles from micrographs
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5i8h

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 17.36 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 21629
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)

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