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- EMDB-23175: nsEM maps of BG505 SOSIP MD39 in complex with the polyclonal Fab ... -

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Basic information

Entry
Database: EMDB / ID: EMD-23175
TitlensEM maps of BG505 SOSIP MD39 in complex with the polyclonal Fab samples from Group 1 of immunized rhesus macaques (Animal IDs: Rh.32034, Rh.32113, Rh.33104, Rh.33395, Rh.34909, Rh.34943)
Map data3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab sample from animal Rh.34943. Obtained through negative stain EM.
Sample
  • Complex: BG505 SOSIP MD39 in complex with the polyclonal Fab samples from Group 1 of immunized rhesus macaques (Animal IDs: Rh.32034, Rh.32113, Rh.33104, Rh.33395, Rh.34909, Rh.34943)
    • Protein or peptide: BG505 SOSIP MD39
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 / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / 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 / Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsAntanasijevic A / Sewall LM / Ward A
Funding support United States, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
CitationJournal: Nat Commun / Year: 2021
Title: Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM.
Authors: Aleksandar Antanasijevic / Leigh M Sewall / Christopher A Cottrell / Diane G Carnathan / Luis E Jimenez / Julia T Ngo / Jennifer B Silverman / Bettina Groschel / Erik Georgeson / Jinal ...Authors: Aleksandar Antanasijevic / Leigh M Sewall / Christopher A Cottrell / Diane G Carnathan / Luis E Jimenez / Julia T Ngo / Jennifer B Silverman / Bettina Groschel / Erik Georgeson / Jinal Bhiman / Raiza Bastidas / Celia LaBranche / Joel D Allen / Jeffrey Copps / Hailee R Perrett / Kimmo Rantalainen / Fabien Cannac / Yuhe R Yang / Alba Torrents de la Peña / Rebeca Froes Rocha / Zachary T Berndsen / David Baker / Neil P King / Rogier W Sanders / John P Moore / Shane Crotty / Max Crispin / David C Montefiori / Dennis R Burton / William R Schief / Guido Silvestri / Andrew B Ward /
Abstract: Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate ...Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.
History
DepositionDec 22, 2020-
Header (metadata) releaseAug 4, 2021-
Map releaseAug 4, 2021-
UpdateNov 23, 2022-
Current statusNov 23, 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
  • Download
  • 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_23175.png

Downloads & links

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Map

FileDownload / File: emd_23175.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab sample from animal Rh.34943. Obtained through negative stain EM.
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.058673333 - 0.133881
Average (Standard dev.)0.00028212988 (±0.006993253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 393.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0590.1340.000

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Supplemental data

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Additional map: 3D map of BG505 SOSIP MD39 in complex...

Fileemd_23175_additional_1.map
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab sample from animal Rh.34909. Obtained through negative stain EM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D map of BG505 SOSIP MD39 in complex...

Fileemd_23175_additional_2.map
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab sample from animal Rh.33395. Obtained through negative stain EM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D map of BG505 SOSIP MD39 in complex...

Fileemd_23175_additional_3.map
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab sample from animal Rh.33104. Obtained through negative stain EM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D map of BG505 SOSIP MD39 in complex...

Fileemd_23175_additional_4.map
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab sample from animal Rh.32113. Obtained through negative stain EM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D map of BG505 SOSIP MD39 in complex...

Fileemd_23175_additional_5.map
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab sample from animal Rh.32034. Obtained through negative stain EM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : BG505 SOSIP MD39 in complex with the polyclonal Fab samples from ...

EntireName: BG505 SOSIP MD39 in complex with the polyclonal Fab samples from Group 1 of immunized rhesus macaques (Animal IDs: Rh.32034, Rh.32113, Rh.33104, Rh.33395, Rh.34909, Rh.34943)
Components
  • Complex: BG505 SOSIP MD39 in complex with the polyclonal Fab samples from Group 1 of immunized rhesus macaques (Animal IDs: Rh.32034, Rh.32113, Rh.33104, Rh.33395, Rh.34909, Rh.34943)
    • Protein or peptide: BG505 SOSIP MD39

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Supramolecule #1: BG505 SOSIP MD39 in complex with the polyclonal Fab samples from ...

SupramoleculeName: BG505 SOSIP MD39 in complex with the polyclonal Fab samples from Group 1 of immunized rhesus macaques (Animal IDs: Rh.32034, Rh.32113, Rh.33104, Rh.33395, Rh.34909, Rh.34943)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Polyclonal complexes were generated by incubation of isolated polyclonal Fabs with recombinantly expressed BG505 SOSIP and subsequent SEC purification.
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F / Recombinant plasmid: pPPI4

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Macromolecule #1: BG505 SOSIP MD39

MacromoleculeName: BG505 SOSIP MD39 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETEKHNV WATHACVPTD PNPQEIHLEN VTEEFNMWKN NMVEQMHEDI ISLWDQSLKP CVKLTPLCVT LQCTNVTNNI TDDMRGELKN CSFNMTTELR DKKQKVYSLF ...String:
MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETEKHNV WATHACVPTD PNPQEIHLEN VTEEFNMWKN NMVEQMHEDI ISLWDQSLKP CVKLTPLCVT LQCTNVTNNI TDDMRGELKN CSFNMTTELR DKKQKVYSLF YRLDVVQINE NQGNRSNNSN KEYRLINCNT SAITQACPKV SFEPIPIHYC APAGWAILKC KDKKFNGTGP CPSVSTVQCT HGIKPVVSTQ LLLNGSLAEE EVIIRSENIT NNAKNILVQF NTPVQINCTR PNNNTVKSIR IGPGQAFYYT GDIIGDIRQA HCNVSKATWN ETLGKVVKQL RKHFGNNTII RFAQSSGGDL EVTTHSFNCG GEFFYCNTSG LFNSTWISNT SVQGSNSTGS NDSITLPCRI KQIINMWQRI GQAMYAPPIQ GVIRCVSNIT GLILTRDGGS TNSTTETFRP GGGDMRDNWR SELYKYKVVK IEPLGVAPTR CKRRVVGRRR RRRAVGIGAV SLGFLGAAGS TMGAASMTLT VQARNLLSGI VQQQSNLLRA PEPQQHLLKD THWGIKQLQA RVLAVEHYLR DQQLLGIWGC SGKLICCTNV PWNSSWSNRN LSEIWDNMTW LQWDKEISNY TQIIYGLLEE SQNQQEKNEQ DLLALD

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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

Concentration0.05 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMTris-HClTris
150.0 mMsodium chlorideNaClSodium chloride

Details: TBS buffer, pH 7.4
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Sample diluted to 0.05 mg/mL. 3 uL was applied onto the grid, blotted off, and then stained with 2% uranyl formate for 60 seconds.
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
DetailsPolyclonal complexes were generated by incubation of isolated polyclonal Fabs with recombinantly expressed BG505 SOSIP and subsequent SEC purification.

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 100.0 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: 52000
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 Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Appion manual picker
Startup modelType of model: OTHER
Details: Negative stain EM map of the free BG505 SOSIP trimer
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 100000

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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