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- PDB-7l8a: BG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-1 from a... -

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Basic information

Entry
Database: PDB / ID: 7l8a
TitleBG505 SOSIP MD39 in complex with the polyclonal Fab pAbC-1 from animal Rh.33104 (Wk26 time point)
Components
  • (BG505 SOSIP MD39 - ...) x 2
  • (Rh.33104 pAbC-1 - ...) x 2
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / HIV / vaccine design / BG505 / VIRAL PROTEIN / Polyclonal antibodies / EMPEM / VIRAL PROTEIN-IMMUNE SYSTEM complex
Biological speciesHuman immunodeficiency virus 1
Macaca mulatta (Rhesus monkey)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAntanasijevic, A. / Sewall, L.M. / Torrents de la Pena, A. / 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 31, 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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Assembly

Deposited unit
E: BG505 SOSIP MD39 - gp120
F: BG505 SOSIP MD39 - gp41
H: Rh.33104 pAbC-1 - Heavy Chain
L: Rh.33104 pAbC-1 - Light Chain
A: BG505 SOSIP MD39 - gp120
B: BG505 SOSIP MD39 - gp41
C: BG505 SOSIP MD39 - gp120
D: BG505 SOSIP MD39 - gp41
hetero molecules


Theoretical massNumber of molelcules
Total (without water)246,10164
Polymers225,7118
Non-polymers20,39056
Water00
1


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

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Components

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BG505 SOSIP MD39 - ... , 2 types, 6 molecules EACFBD

#1: Protein BG505 SOSIP MD39 - gp120


Mass: 52532.418 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 MD39 - gp41


Mass: 16451.553 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)

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Rh.33104 pAbC-1 - ... , 2 types, 2 molecules HL

#3: Protein Rh.33104 pAbC-1 - Heavy Chain


Mass: 9805.078 Da / Num. of mol.: 1 / Source method: isolated from a natural source
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.
Source: (natural) Macaca mulatta (Rhesus monkey)
#4: Protein Rh.33104 pAbC-1 - Light Chain


Mass: 8954.028 Da / Num. of mol.: 1 / Source method: isolated from a natural source
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.
Source: (natural) Macaca mulatta (Rhesus monkey)

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Sugars , 4 types, 56 molecules

#5: 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.: 30
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#7: 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.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#8: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 23 / 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

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Details

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 MD39 in complex with the polyclonal Fab pAbC-1 from animal Rh.33104 (Wk26 time point)
Type: COMPLEX
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.
Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Human immunodeficiency virus 111676
21Macaca mulatta (Rhesus monkey)9544
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4 / Details: TBS buffer prepared from a 10X stock
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTris-HCl1
2150 mMSodium chlorideNaCl1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Polyclonal complexes were generated by incubation of isolated polyclonal Fabs with recombinantly expressed BG505 SOSIP and subsequent SEC purification.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 7.5 sec. / Electron dose: 44.9 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3194
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4RELION3CTF correction
7UCSF Chimeramodel fitting
9Cootmodel refinement
10RosettaEMmodel refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
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: 502068 / Details: Particles picked using template picker
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81561 / Algorithm: BACK PROJECTION
Details: 81561 symmetry-expanded particles. See the Method section of the manuscript for details.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
16VL5

6vl5

1
24KTE

4kte

1

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