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- PDB-7mep: BG505 SOSIP.v5.2(7S) in complex with the monoclonal antibodies Rh... -

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Basic information

Entry
Database: PDB / ID: 7mep
TitleBG505 SOSIP.v5.2(7S) in complex with the monoclonal antibodies Rh.33172 mAb.1 and RM19R
Components
  • (Rh.33172 mAb.1 ...) x 2
  • BG505 SOSIPv5.2(7S) - gp120
  • RM19R mAb Heavy chain
  • RM19R mAb Light chain
KeywordsVIRAL PROTEIN / Immune complex / monoclonal antibodies / HIV-1 / BG505 SOSIP
Function / homologyImmunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Function and homology information
Biological speciesMacaca mulatta (Rhesus monkey)
Human immunodeficiency virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsAntanasijevic, A. / Ward, A.B.
Funding support United States, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782, OPP1196345 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 7, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 10, 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
Revision 1.2Nov 13, 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
G: RM19R mAb Light chain
I: RM19R mAb Heavy chain
A: BG505 SOSIPv5.2(7S) - gp120
B: BG505 SOSIPv5.2(7S) - gp120
H: Rh.33172 mAb.1 Heavy chain
L: Rh.33172 mAb.1 Light chain
J: RM19R mAb Light chain
M: RM19R mAb Heavy chain
C: BG505 SOSIPv5.2(7S) - gp120
E: BG505 SOSIPv5.2(7S) - gp120
K: RM19R mAb Light chain
N: RM19R mAb Heavy chain
D: BG505 SOSIPv5.2(7S) - gp120
F: BG505 SOSIPv5.2(7S) - gp120
hetero molecules


Theoretical massNumber of molelcules
Total (without water)570,04580
Polymers550,93814
Non-polymers19,10766
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 6 molecules ABCEDF

#3: Protein
BG505 SOSIPv5.2(7S) - gp120


Mass: 74987.219 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus / Plasmid: pPPI4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)

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Antibody , 4 types, 8 molecules GJKIMNHL

#1: Antibody RM19R mAb Light chain


Mass: 11808.134 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Plasmid: pPPI4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Antibody RM19R mAb Heavy chain


Mass: 13268.667 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Plasmid: pPPI4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#4: Antibody Rh.33172 mAb.1 Heavy chain


Mass: 14306.693 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Plasmid: pcDNA3.4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#5: Antibody Rh.33172 mAb.1 Light chain


Mass: 11477.829 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Plasmid: pcDNA3.4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)

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Sugars , 3 types, 66 molecules

#6: 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
#7: 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.: 12
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
#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.: 51 / 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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1BG505 SOSIP.v5.2(7S) in complex with the monoclonal antibodies Rh.33172 mAb.1 and RM19RCOMPLEXThe complexes were created by combining BG505 SOSIP.v5.2(7S) and the two antibodies (as Fab fragments) and subsequent SEC purification.#1-#50MULTIPLE SOURCES
2monoclonal antibodies Rh.33172 mAb.1 and RM19RCOMPLEX#1-#2, #4-#51RECOMBINANT
3BG505 SOSIP.v5.2(7S)COMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Macaca mulatta (Rhesus monkey)9544
23Human immunodeficiency virus12721
Buffer solutionpH: 7.4 / Details: TBS, 0.2um filtered
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris-HClTris-HCl1
2150 mMSodium ChlorideNaCl1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse.
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: Blot force: 0 Wait time: 10s Blot time: 3-7s

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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: 700 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: 9.5 sec. / Electron dose: 44.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2050
Image scansMovie frames/image: 38

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
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: 169887 / Details: template picked
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35099 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6VFL

6vfl


Accession code: 6VFL / Source name: PDB / Type: experimental model

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