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- EMDB-23222: Designed tetrahedral nanoparticle T33-31 presenting BG505 SOSIP t... -

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Basic information

Entry
Database: EMDB / ID: EMD-23222
TitleDesigned tetrahedral nanoparticle T33-31 presenting BG505 SOSIP trimers
Map dataBG505 SOSIP-T33-31 nanoparticle - Main map
Sample
  • Complex: Designed tetrahedral nanoparticle BG505 SOSIP-T33-31
    • Protein or peptide: BG505 SOSIP-T33-31B
    • Protein or peptide: BG505 SOSIP-T33-31A
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 / cryo EM / Resolution: 2.9 Å
AuthorsAntanasijevic A / Sewall LM / 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
DepositionDec 31, 2020-
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: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7l85
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23222.png

Downloads & links

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Map

FileDownload / File: emd_23222.map.gz / Format: CCP4 / Size: 536.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBG505 SOSIP-T33-31 nanoparticle - Main map
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.045
Minimum - Maximum-0.12806676 - 0.22880295
Average (Standard dev.)-2.860701e-05 (±0.0035960267)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions520520520
Spacing520520520
CellA=B=C: 598.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z520520520
origin x/y/z0.0000.0000.000
length x/y/z598.000598.000598.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS520520520
D min/max/mean-0.1280.229-0.000

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Supplemental data

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Mask #1

Fileemd_23222_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BG505 SOSIP-T33-31 nanoparticle - Half-map 1

Fileemd_23222_half_map_1.map
AnnotationBG505 SOSIP-T33-31 nanoparticle - Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BG505 SOSIP-T33-31 nanoparticle - Half-map 2

Fileemd_23222_half_map_2.map
AnnotationBG505 SOSIP-T33-31 nanoparticle - 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 : Designed tetrahedral nanoparticle BG505 SOSIP-T33-31

EntireName: Designed tetrahedral nanoparticle BG505 SOSIP-T33-31
Components
  • Complex: Designed tetrahedral nanoparticle BG505 SOSIP-T33-31
    • Protein or peptide: BG505 SOSIP-T33-31B
    • Protein or peptide: BG505 SOSIP-T33-31A

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Supramolecule #1: Designed tetrahedral nanoparticle BG505 SOSIP-T33-31

SupramoleculeName: Designed tetrahedral nanoparticle BG505 SOSIP-T33-31 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The map was generated by focused refinement of BG505 SOSIP-T33-31 nanoparticle dataset using a mask around the T33-31 nanoparticle core (masking out the flexibly linked antigens).
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 / Recombinant plasmid: pPPI4

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Macromolecule #1: BG505 SOSIP-T33-31B

MacromoleculeName: BG505 SOSIP-T33-31B / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 13.679698 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GSVRGIRGAI TVEEDTPAAI LAATIELLLK MLEANGIQSY EELAAVIFTV TEDLTSAFPA EAARLIGMHR VPLLSAREVP VPGSLPRVI RVLALWNTDT PQDRVRHVYL NEAVRLRPDL ESAQLE

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Macromolecule #2: BG505 SOSIP-T33-31A

MacromoleculeName: BG505 SOSIP-T33-31A / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 11.698429 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
GGEEVVLITV PSALVAVKIA HALVEERLAA CVNIVPGLTS IYREEGSVVS DHELLLLVKT TTDAFPKLKE RVKELHPYEV PEIVALPIA EGNREYLDWL RENTGLE

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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

Concentration4.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMTris-HClTris
150.0 mMSodium chlorideNaClSodium chloride

Details: TBS buffer prepared from a 10X stock
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / 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..
DetailsBG505 SOSIP-T33-31 nanoparticle was prepared by combining equimolar amounts of BG505 SOSIP-T33-31A and BG505 SOSIP-T33-31B components that were expressed separately.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
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: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
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: 1-41 / Number grids imaged: 2 / Number real images: 1751 / Average exposure time: 10.25 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 223099 / Details: Gaussian picker in Relion/3.0
CTF correctionSoftware - Name: RELION (ver. 3.0)
Startup modelType of model: OTHER
Details: Map obtained from Ab initio reconstruction in cryoSPARC with application of symmetry
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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: T (tetrahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 110369
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 modelPDB ID:

4zk7

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7l85:
Designed tetrahedral nanoparticle T33-31 presenting BG505 SOSIP trimers

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