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- EMDB-23778: Negative stain EM map of BG505 SOSIP in complex with Rh4O9.8 mono... -

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Basic information

Entry
Database: EMDB / ID: EMD-23778
TitleNegative stain EM map of BG505 SOSIP in complex with Rh4O9.8 monoclonal antibody (as Fab fragment)
Map data3D map of BG505 SOSIP in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment). Obtained by negative stain EM.
Sample
  • Complex: BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment)
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / structural molecule activity / virion attachment to host cell / host cell plasma membrane / virion membrane / identical protein binding / plasma membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus
Methodsingle particle reconstruction / negative staining / Resolution: 15.9 Å
AuthorsAntanasijevic A / Ward AB
Funding support United States, 2 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
Bill & Melinda Gates FoundationOPP1196345 United States
CitationJournal: Sci Adv / Year: 2022
Title: From structure to sequence: Antibody discovery using cryoEM.
Authors: Aleksandar Antanasijevic / Charles A Bowman / Robert N Kirchdoerfer / Christopher A Cottrell / Gabriel Ozorowski / Amit A Upadhyay / Kimberly M Cirelli / Diane G Carnathan / Chiamaka A ...Authors: Aleksandar Antanasijevic / Charles A Bowman / Robert N Kirchdoerfer / Christopher A Cottrell / Gabriel Ozorowski / Amit A Upadhyay / Kimberly M Cirelli / Diane G Carnathan / Chiamaka A Enemuo / Leigh M Sewall / Bartek Nogal / Fangzhu Zhao / Bettina Groschel / William R Schief / Devin Sok / Guido Silvestri / Shane Crotty / Steven E Bosinger / Andrew B Ward /
Abstract: One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and ...One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.
History
DepositionApr 6, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23778.png

Downloads & links

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Map

FileDownload / File: emd_23778.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of BG505 SOSIP in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment). Obtained by negative stain EM.
Voxel sizeX=Y=Z: 1.77 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.034227178 - 0.058464553
Average (Standard dev.)-7.9607256e-05 (±0.0033659055)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 396.47998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z396.480396.480396.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0340.058-0.000

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Supplemental data

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Sample components

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Entire : BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 ...

EntireName: BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment)
Components
  • Complex: BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment)

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Supramolecule #1: BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 ...

SupramoleculeName: BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment)
type: complex / ID: 1 / Parent: 0
Details: Fab fragment was generated by proteolytic cleavage of IgG antibody
Source (natural)Organism: Human immunodeficiency virus
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F / Recombinant plasmid: pPPI4

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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:
ConcentrationFormulaName
10.0 mMTris-HClTrisTris-HClTris
150.0 mMNaClSodium chlorideSodium chloride

Details: TBS, 0.2um filtered
StainingType: NEGATIVE / Material: Uranyl formate / Details: The samples were stained with UF for 60s.
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE
DetailsThe complex was generated by incubation of Rh4O9.8 monoclonal antibody (as Fab) with recombinantly expressed BG505 SOSIP and subsequent SEC purification.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 85277 / Details: DoG Picker in Appion
Startup modelType of model: OTHER / Details: Low-pass-filtered map of BG505 SOSIP
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: Relion, regularized likelihood
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: Relion, regularized likelihood
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 15.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 9525

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