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- EMDB-7555: CryoEM map of BG505 SOSIP.664 in complex with post boost 1 serum ... -

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Basic information

Entry
Database: EMDB / ID: EMD-7555
TitleCryoEM map of BG505 SOSIP.664 in complex with post boost 1 serum from rabbit 3417
Map dataBG505 SOSIP.664 in complex with serum from rabbit 3417 post boost 1. Map of antibodies bound to trimer after several rounds of 3D classification in CryoSparc.
Sample
  • Complex: Complex of BG505 SOSIP.664 and serum antibodies from post boost 1 from rabbit 3417. Subset of particles from dataset.
Biological speciesHuman immunodeficiency virus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsTurner HL / Ward AB / Nogal B
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
DepositionMar 13, 2018-
Header (metadata) releaseApr 18, 2018-
Map releaseSep 5, 2018-
UpdateMay 15, 2019-
Current statusMay 15, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.381
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.381
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7555.png

Downloads & links

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Map

FileDownload / File: emd_7555.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBG505 SOSIP.664 in complex with serum from rabbit 3417 post boost 1. Map of antibodies bound to trimer after several rounds of 3D classification in CryoSparc.
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.381 / Movie #1: 0.381
Minimum - Maximum-0.31383744 - 1.1800797
Average (Standard dev.)0.0032141434 (±0.061049055)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 345.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z345.000345.000345.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ208208208
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.3141.1800.003

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Supplemental data

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Sample components

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Entire : Complex of BG505 SOSIP.664 and serum antibodies from post boost 1...

EntireName: Complex of BG505 SOSIP.664 and serum antibodies from post boost 1 from rabbit 3417. Subset of particles from dataset.
Components
  • Complex: Complex of BG505 SOSIP.664 and serum antibodies from post boost 1 from rabbit 3417. Subset of particles from dataset.

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Supramolecule #1: Complex of BG505 SOSIP.664 and serum antibodies from post boost 1...

SupramoleculeName: Complex of BG505 SOSIP.664 and serum antibodies from post boost 1 from rabbit 3417. Subset of particles from dataset.
type: complex / ID: 1 / Parent: 0
Details: Serum was purified through SEC after using a Protein A or G column to capture complex then cleave IgG releasing Fab and trimer. Substoichiometric occupancy and flexibility show lack of ...Details: Serum was purified through SEC after using a Protein A or G column to capture complex then cleave IgG releasing Fab and trimer. Substoichiometric occupancy and flexibility show lack of density in Fab region while trimer core is refined to higher resolution. Map shows strong density of single Fab bound to glycan hole and low densities of Fabs bound to the trimer base.
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

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.44 mg/mL
BufferpH: 7.4 / Details: TBS
GridModel: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsSample in TBS, added 0.5uL of DDM at 0.42mM. Sample was monodisperse

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-46 / Average exposure time: 11.5 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / 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

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.6.3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.6.3) / Number images used: 14274

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