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- PDB-7l7t: BG505 SOSIP reconstructed from a designed nanoparticle, BG505 SOS... -

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Basic information

Entry
Database: PDB / ID: 7l7t
TitleBG505 SOSIP reconstructed from a designed nanoparticle, BG505 SOSIP-T33-31 (Component A)
Components
  • BG505 SOSIP.v5.2(7S) - gp120
  • BG505 SOSIP.v5.2(7S) - gp41
KeywordsVIRAL PROTEIN / HIV / vaccine design / protein design / nanoparticles / BG505
Biological speciesHuman immunodeficiency virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsAntanasijevic, A. / Cannac, F. / Sewall, L.M. / Ward, A.B.
Funding support United States, 1items
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 30, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 4, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 25, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • EMDB-23218
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Assembly

Deposited unit
A: BG505 SOSIP.v5.2(7S) - gp120
B: BG505 SOSIP.v5.2(7S) - gp41
C: BG505 SOSIP.v5.2(7S) - gp120
D: BG505 SOSIP.v5.2(7S) - gp41
E: BG505 SOSIP.v5.2(7S) - gp120
F: BG505 SOSIP.v5.2(7S) - gp41
hetero molecules


Theoretical massNumber of molelcules
Total (without water)241,29972
Polymers219,7536
Non-polymers21,54566
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein BG505 SOSIP.v5.2(7S) - gp120


Mass: 56707.480 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Plasmid: pPPI4 / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#2: Protein BG505 SOSIP.v5.2(7S) - gp41


Mass: 16543.627 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Plasmid: pPPI4 / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#3: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#4: Polysaccharide...
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 39 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: BG505 SOSIP reconstructed from a designed nanoparticle, BG505 SOSIP-T33-31 (Component A)
Type: COMPLEX
Details: The map was generated by subparticle extraction from the BG505 SOSIP-T33-31 nanoparticle using the localized reconstruction approach.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Human immunodeficiency virus 1
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: pPPI4
Buffer solutionpH: 7.4 / Details: TBS buffer prepared from a 10X stock
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTris1
2150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 4.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: BG505 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.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K / Details: Blotting time varied between 3 and 7 seconds.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 10.25 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1751
Image scansMovie frames/image: 41 / Used frames/image: 1-41

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2Leginonimage acquisition
4RELION3CTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13Cootmodel refinement
14RosettaEMmodel refinement
Image processingDetails: Frames were aligned using MotionCorr and GCTF was applied for estimation of CTF parameters.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 441476
Details: Subparticle were generated by extraction from 110,369 BG505 SOSIP-T33-31 nanoparticles (4 subparticles per 1 nanoparticle).
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106478 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6VL5

6vl5

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