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- PDB-7mdu: BG505 SOSIP MD39 in complex with the monoclonal antibodies Rh.331... -

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Basic information

Entry
Database: PDB / ID: 7mdu
TitleBG505 SOSIP MD39 in complex with the monoclonal antibodies Rh.33104 mAb.1 and RM20A3
Components
  • (Rh.33104 mAb.1 ...) x 2
  • RM20A3 mAb Heavy Chain
  • RM20A3 mAb Light Chain
  • Surface protein gp120
  • Transmembrane protein gp41
KeywordsVIRAL PROTEIN / monoclonal antibody / immune complex
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / 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 ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / 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 / 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 / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesMacaca mulatta (Rhesus monkey)
Human immunodeficiency virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAntanasijevic, A. / 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

Movie
  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-23780
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  • Superimposition on EM map
  • EMDB-23780
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
H: Rh.33104 mAb.1 Heavy Chain
A: Surface protein gp120
E: RM20A3 mAb Heavy Chain
F: RM20A3 mAb Light Chain
L: Rh.33104 mAb.1 Light Chain
B: Transmembrane protein gp41
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,95827
Polymers123,9976
Non-polymers6,96121
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18350 Å2
ΔGint53 kcal/mol
Surface area50640 Å2

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 57652.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Plasmid: pPPI4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q2N0S6
#6: Protein Transmembrane protein gp41 / TM / Glycoprotein 41 / gp41


Mass: 17134.324 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Plasmid: pPPI4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q2N0S6

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Antibody , 4 types, 4 molecules HEFL

#1: Antibody Rh.33104 mAb.1 Heavy Chain


Mass: 12421.753 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)
#3: Antibody RM20A3 mAb Heavy Chain


Mass: 13511.111 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)
#4: Antibody RM20A3 mAb Light Chain


Mass: 11755.821 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.33104 mAb.1 Light Chain


Mass: 11521.576 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, 21 molecules

#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.: 8
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
#8: 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.: 1
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
#9: 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.: 12 / 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 MD39 in complex with the monoclonal antibodies Rh.33104 mAb.1 and RM20A3
Type: COMPLEX
Details: The complexes were created by combining BG505 SOSIP MD39 and the two antibodies (as Fab fragments) and subsequent SEC purification.
Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4 / Details: TBS, 0.2um filtered
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTrisTris-HCl1
2150 mMSodium ChlorideNaCl1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The specimen consisted of homogeneous antigen-antibody complexes
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: 800 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: 1022
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: 109105 / Details: template picking
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29009 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16VFL

6vfl

16VFL1PDBexperimental model
26X9R

6x9r

16X9R2PDBexperimental model

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