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- EMDB-23240: BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAb... -

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Basic information

Entry
Database: EMDB / ID: EMD-23240
TitleBG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point)
Map data3D map of BG505 SOSIP v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point). Main map
Sample
  • Complex: BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point)
    • Complex: polyclonal Fab pAbC-5 from animal Rh.33311
      • Protein or peptide: Rh.33311 pAbC-5 - Heavy Chain
      • Protein or peptide: Rh.33311 pAbC-5 - Light Chain
    • Complex: BG505 SOSIP.v5.2 N241/N289 gp120/gp41
      • Protein or peptide: BG505 SOSIP.v5.2 N241/N289 - gp120
      • Protein or peptide: BG505 SOSIP.v5.2 N241/N289 - gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 speciesMacaca mulatta (Rhesus monkey) / Human immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsAntanasijevic A / Sewall LM / Cannac F / Ward AB
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
DepositionJan 1, 2021-
Header (metadata) releaseAug 4, 2021-
Map releaseAug 4, 2021-
UpdateAug 25, 2021-
Current statusAug 25, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7l8y
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7l8y
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23240.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of BG505 SOSIP v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point). Main map
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.022
Minimum - Maximum-0.05761562 - 0.09345373
Average (Standard dev.)0.00015761724 (±0.0028721131)
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-0.0580.0930.000

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Supplemental data

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Mask #1

Fileemd_23240_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: 3D map of BG505 SOSIP v5.2 N241/N289 in...

Fileemd_23240_half_map_1.map
Annotation3D map of BG505 SOSIP v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point). Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: 3D map of BG505 SOSIP v5.2 N241/N289 in...

Fileemd_23240_half_map_2.map
Annotation3D map of BG505 SOSIP v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point). Half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAb...

EntireName: BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point)
Components
  • Complex: BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point)
    • Complex: polyclonal Fab pAbC-5 from animal Rh.33311
      • Protein or peptide: Rh.33311 pAbC-5 - Heavy Chain
      • Protein or peptide: Rh.33311 pAbC-5 - Light Chain
    • Complex: BG505 SOSIP.v5.2 N241/N289 gp120/gp41
      • Protein or peptide: BG505 SOSIP.v5.2 N241/N289 - gp120
      • Protein or peptide: BG505 SOSIP.v5.2 N241/N289 - gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAb...

SupramoleculeName: BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (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.

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Supramolecule #2: polyclonal Fab pAbC-5 from animal Rh.33311

SupramoleculeName: polyclonal Fab pAbC-5 from animal Rh.33311 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Macaca mulatta (Rhesus monkey)

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Supramolecule #3: BG505 SOSIP.v5.2 N241/N289 gp120/gp41

SupramoleculeName: BG505 SOSIP.v5.2 N241/N289 gp120/gp41 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Rh.33311 pAbC-5 - Heavy Chain

MacromoleculeName: Rh.33311 pAbC-5 - Heavy Chain / type: protein_or_peptide / ID: 1 / 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 #2: Rh.33311 pAbC-5 - Light Chain

MacromoleculeName: Rh.33311 pAbC-5 - Light Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Molecular weightTheoretical: 8.443399 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)

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Macromolecule #3: BG505 SOSIP.v5.2 N241/N289 - gp120

MacromoleculeName: BG505 SOSIP.v5.2 N241/N289 - gp120 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 56.417125 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS ...String:
MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS LFYRLDVVQI NENQGNRSNN SNKEYRLINC NTSAITQACP KVSFEPIPIH YCAPAGFAIL KCKDKKFNGT GP CTNVSTV QCTHGIKPVV STQLLLNGSL AEEEVIIRSE NITNNAKNIL VQLNESVQIN CTRPNNNTRK SIRIGPGQWF YAT GDIIGD IRQAHCNVSK ATWNETLGKV VKQLRKHFGN NTIIRFANSS GGDLEVTTHS FNCGGEFFYC NTSGLFNSTW ISNT SVQGS NSTGSNDSIT LPCRIKQIIN MWQRIGQAMY APPIQGVIRC VSNITGLILT RDGGSTNSTT ETFRPGGGDM RDNWR SELY KYKVVKIEPL GVAPTRCKR

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Macromolecule #4: BG505 SOSIP.v5.2 N241/N289 - gp41

MacromoleculeName: BG505 SOSIP.v5.2 N241/N289 - gp41 / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 16.477719 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
LGFLGAAGST MGAASMTLTV QARNLLSGIV QQQSNLLRAP ECQQHLLKLT VWGIKQLQAR VLAVERYLRD QQLLGIWGCS GKLICCTNV PWNSTWSNRN LSEIWDNMTW LQWDKEISNY TQIIYGLLEE SQNQQEKNEQ DLLALD

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 45 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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-HClTrisTris-HClTris
150.0 mMNaClSodium chlorideSodium 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 - 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
Electron beamAcceleration voltage: 300 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: 1.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-39 / Number grids imaged: 4 / Number real images: 6268 / Average exposure time: 9.75 sec. / Average electron dose: 44.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1267693 / Details: Particles picked using template picker
CTF correctionSoftware - Name: RELION (ver. 3.0)
Startup modelType of model: OTHER
Details: 3D map of the free HIV Env trimer ectodomain, low-pass filtered to 20A
Initial 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
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: 52082 symmetry-expanded particles. See the Methods section of the manuscript for details.
Number images used: 52082
DetailsFrames were aligned using MotionCorr and GCTF was applied for estimation of CTF parameters.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:
,
)
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7l8y:
BG505 SOSIP.v5.2 N241/N289 in complex with the polyclonal Fab pAbC-5 from animal Rh.33311 (Wk26 time point)

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