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- EMDB-23229: BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from a... -

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Basic information

Entry
Database: EMDB / ID: EMD-23229
TitleBG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point)
Map data3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point). Main map
Sample
  • Complex: BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point)
    • Protein or peptide: BG505 SOSIP MD39 - gp120
    • Protein or peptide: BG505 SOSIP MD39 - gp41
    • Protein or peptide: Rh.33104 pAbC-3 - Heavy Chain
    • Protein or peptide: Rh.33104 pAbC-3 - Light Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsHIV / vaccine design / BG505 / VIRAL PROTEIN / Polyclonal antibodies / EMPEM / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / 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 ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / 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 / 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 / Macaca mulatta (Rhesus monkey)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAntanasijevic A / Sewall LM
Funding support United States, 1 items
OrganizationGrant numberCountry
International AIDS Vaccine InitiativeOPP1196345 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 31, 2020-
Header (metadata) releaseAug 4, 2021-
Map releaseAug 4, 2021-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7l8c
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7l8c
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23229.png

Downloads & links

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Map

FileDownload / File: emd_23229.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point). Main map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 360 pix.
= 370.8 Å		1.03 Å/pix.
x 360 pix.
= 370.8 Å		1.03 Å/pix.
x 360 pix.
= 370.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.025
Minimum - Maximum-0.07060832 - 0.13361056
Average (Standard dev.)0.000081402046 (±0.0037824512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 370.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z370.800370.800370.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0710.1340.000

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Supplemental data

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Mask #1

Fileemd_23229_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_23229_half_map_1.map
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point). Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_23229_half_map_2.map
Annotation3D map of BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point). Half-map 2
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 pAbC-3 from a...

EntireName: BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point)
Components
  • Complex: BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point)
    • Protein or peptide: BG505 SOSIP MD39 - gp120
    • Protein or peptide: BG505 SOSIP MD39 - gp41
    • Protein or peptide: Rh.33104 pAbC-3 - Heavy Chain
    • Protein or peptide: Rh.33104 pAbC-3 - Light Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from a...

SupramoleculeName: BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Polyclonal complexes were generated by incubation of isolated polyclonal Fabs with recombinantly expressed BG505 SOSIP and subsequent SEC purification. Map and model were reconstructed from ...Details: Polyclonal complexes were generated by incubation of isolated polyclonal Fabs with recombinantly expressed BG505 SOSIP and subsequent SEC purification. Map and model were reconstructed from the immune complex dataset using the focused classification approach.
Source (natural)Organism: Human immunodeficiency virus 1

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Macromolecule #1: BG505 SOSIP MD39 - gp120

MacromoleculeName: BG505 SOSIP MD39 - gp120 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 52.532418 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: NLWVTVYYGV PVWKDAETTL FCASDAKAYE TEKHNVWATH ACVPTDPNPQ EIHLENVTEE FNMWKNNMVE QMHEDIISLW DQSLKPCVK LTPLCVTLQC TNVTNNITDD MRGELKNCSF NMTTELRDKK QKVYSLFYRL DVVQINENQG NRSNNSNKEY R LINCNTSA ...String:
NLWVTVYYGV PVWKDAETTL FCASDAKAYE TEKHNVWATH ACVPTDPNPQ EIHLENVTEE FNMWKNNMVE QMHEDIISLW DQSLKPCVK LTPLCVTLQC TNVTNNITDD MRGELKNCSF NMTTELRDKK QKVYSLFYRL DVVQINENQG NRSNNSNKEY R LINCNTSA ITQACPKVSF EPIPIHYCAP AGFAILKCKD KKFNGTGPCP SVSTVQCTHG IKPVVSTQLL LNGSLAEEEV II RSENITN NAKNILVQLN TPVQINCTRP NNNTVKSIRI GPGQAFYYTG DIIGDIRQAH CNVSKATWNE TLGKVVKQLR KHF GNNTII RFAQSSGGDL EVTTHSFNCG GEFFYCNTSG LFNSTWISNT SVQGSNSTGS NDSITLPCRI KQIINMWQRI GQAM YAPPI QGVIRCVSNI TGLILTRDGG STNSTTETFR PGGGDMRDNW RSELYKYKVV KIEPLGVAPT RCKR

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Macromolecule #2: BG505 SOSIP MD39 - gp41

MacromoleculeName: BG505 SOSIP MD39 - gp41 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 16.665773 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
VSLGFLGAAG STMGAASMTL TVQARNLLSG IVQQQSNLLR APEPQQHLLK DTHWGIKQLQ ARVLAVEHYL RDQQLLGIWG CSGKLICCT NVPWNSSWSN RNLSEIWDNM TWLQWDKEIS NYTQIIYGLL EESQNQQEKN EQDLLALD

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Macromolecule #3: Rh.33104 pAbC-3 - Heavy Chain

MacromoleculeName: Rh.33104 pAbC-3 - Heavy Chain / type: protein_or_peptide / ID: 3
Details: This is a polyclonal antibody and the sequence is inherently unknown. Poly-alanine pseudo-model is used to depict the backbone of this antibody.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 9.634868 KDa
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)

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Macromolecule #4: Rh.33104 pAbC-3 - Light Chain

MacromoleculeName: Rh.33104 pAbC-3 - Light Chain / type: protein_or_peptide / ID: 4
Details: This is a polyclonal antibody and the sequence is inherently unknown. Poly-alanine pseudo-model is used to depict the backbone of this antibody.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 9.124238 KDa
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 17 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris-HCl
150.0 mMNaClSodium chloride

Details: TBS buffer prepared from a 10X stock
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time varied between 3 and 7 seconds..
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 TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 2 / Number real images: 3194 / Average exposure time: 7.5 sec. / Average electron dose: 44.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFrames were aligned using MotionCorr and GCTF was applied for estimation of CTF parameters.
Particle selectionNumber selected: 502068 / Details: Particles picked using template picker
Startup modelType of model: OTHER
Details: 3D map of the free HIV Env trimer ectodomain, low-pass filtered to 20A
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: 58378 symmetry-expanded particles. See the Methods section of the manuscript for details.
Number images used: 58378
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Details: Multi-step focused classification was applied to isolate the particles featuring this type fo polyclonal antibody. See the Method section of the manuscript for details
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6vl5

source_name: PDB, initial_model_type: experimental model

4kte

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7l8c:
BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-3 from animal Rh.33104 (Wk26 time point)

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