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- PDB-7mdt: BG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 ... -

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Basic information

Entry
Database: PDB / ID: 7mdt
TitleBG505 SOSIP.v5.2 in complex with the monoclonal antibody Rh4O9.8 (as Fab fragment)
Components
  • (Rh4O9.8 monoclonal antibody ...) x 2
  • Surface protein gp120
  • Transmembrane protein gp41
KeywordsVIRAL PROTEIN / monoclonal antibody / polyclonal antibody / immune complex
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 / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / 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 / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / apoptotic process / host cell plasma membrane / structural molecule activity / virion membrane / 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
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
Macaca mulatta (Rhesus monkey)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsAntanasijevic, A. / Ozorowski, G. / Nogal, B. / Ward, A.B.
Funding support United States, 2items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
Bill & Melinda Gates FoundationOPP1196345 United States
CitationJournal: Sci Adv / Year: 2022
Title: From structure to sequence: Antibody discovery using cryoEM.
Authors: Aleksandar Antanasijevic / Charles A Bowman / Robert N Kirchdoerfer / Christopher A Cottrell / Gabriel Ozorowski / Amit A Upadhyay / Kimberly M Cirelli / Diane G Carnathan / Chiamaka A ...Authors: Aleksandar Antanasijevic / Charles A Bowman / Robert N Kirchdoerfer / Christopher A Cottrell / Gabriel Ozorowski / Amit A Upadhyay / Kimberly M Cirelli / Diane G Carnathan / Chiamaka A Enemuo / Leigh M Sewall / Bartek Nogal / Fangzhu Zhao / Bettina Groschel / William R Schief / Devin Sok / Guido Silvestri / Shane Crotty / Steven E Bosinger / Andrew B Ward /
Abstract: One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and ...One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.
History
DepositionApr 6, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Database references / Category: citation / citation_author
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 / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 6, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Surface protein gp120
B: Transmembrane protein gp41
L: Rh4O9.8 monoclonal antibody Light Chain
H: Rh4O9.8 monoclonal antibody Heavy Chain
C: Surface protein gp120
E: Surface protein gp120
D: Transmembrane protein gp41
F: Transmembrane protein gp41
hetero molecules


Theoretical massNumber of molelcules
Total (without water)265,41959
Polymers248,9398
Non-polymers16,48051
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47430 Å2
ΔGint114 kcal/mol
Surface area89150 Å2

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Components

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Protein , 2 types, 6 molecules ACEBDF

#1: Protein Surface protein gp120 / SU / Glycoprotein 120 / gp120


Mass: 57775.883 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Plasmid: pPPI4 / Cell (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q2N0S6
#2: Protein Transmembrane protein gp41 / TM / Glycoprotein 41 / gp41


Mass: 17178.549 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Plasmid: pPPI4 / Cell (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q2N0S6

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Antibody , 2 types, 2 molecules LH

#3: Antibody Rh4O9.8 monoclonal antibody Light Chain


Mass: 11339.304 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Plasmid: pPPI4 / Cell (production host): HEK293F / Production host: Homo sapiens (human)
#4: Antibody Rh4O9.8 monoclonal antibody Heavy Chain


Mass: 12736.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Plasmid: pPPI4 / Cell (production host): HEK293F / Production host: Homo sapiens (human)

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Sugars , 4 types, 51 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.: 16
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 beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#7: 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.: 3
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
#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.: 31 / 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
Has protein modificationY

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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.v5.2 in complex with the polyclonal Fab sample isolated from animal Rh4O9.
Type: COMPLEX
Details: The map was generated from polyclonal Fab sample isolated from animal Rh4O9. The polyclonal Fabs were complexed with BG505 SOSIP.v5.2 and imaged. Rh4O9.8 monoclonal antibody sequence was ...Details: The map was generated from polyclonal Fab sample isolated from animal Rh4O9. The polyclonal Fabs were complexed with BG505 SOSIP.v5.2 and imaged. Rh4O9.8 monoclonal antibody sequence was relaxed into the Fab-corresponding density.
Entity ID: #3-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Human immunodeficiency virus
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pPPI4
Buffer solutionpH: 7.4 / Details: TBS, 0.2um filtered
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris-HClTris-HCl1
2150 mMSodium chlorideNaCl1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The complex was generated by incubation of Rh4O9 polyclonal antibody (as Fab) with recombinantly expressed BG505 SOSIP.v5.2 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

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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: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 13.5 sec. / Electron dose: 61 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 54

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13RosettaEMmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 327121 / Details: Template picker in cryoSPARC
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98122 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5CEZ

5cez


Accession code: 5CEZ / Source name: PDB / Type: experimental model

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