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- PDB-9dpe: CryoEM Structure of Human BTN2A1 ectodomain in complex with TCR-b... -

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Basic information

Entry
Database: PDB / ID: 9dpe
TitleCryoEM Structure of Human BTN2A1 ectodomain in complex with TCR-blocking 2A1.12 Fab
Components
  • Butyrophilin subfamily 2 member A1
  • Human IgG1 Fragment Antibody Heavy Chain
  • Human IgG1 Fragment Antibody Light Chain
  • Human variable heavy-chain domain Nanobody
KeywordsIMMUNE SYSTEM / Complex / Antibody / Immune Recognition / SIGNALING PROTEIN
Function / homology
Function and homology information


Butyrophilin (BTN) family interactions / regulation of cytokine production / lipid metabolic process / T cell receptor signaling pathway / signaling receptor binding / external side of plasma membrane / plasma membrane
Similarity search - Function
Butyrophilin subfamily 1/2, SPRY/PRY domain / : / : / Butyrophilin subfamily 3 member A2-like, Ig-C domain / SPRY-associated domain / SPRY-associated / PRY / Butyrophylin-like, SPRY domain / SPRY domain / B30.2/SPRY domain ...Butyrophilin subfamily 1/2, SPRY/PRY domain / : / : / Butyrophilin subfamily 3 member A2-like, Ig-C domain / SPRY-associated domain / SPRY-associated / PRY / Butyrophylin-like, SPRY domain / SPRY domain / B30.2/SPRY domain / B30.2/SPRY domain profile. / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / SPRY domain / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Concanavalin A-like lectin/glucanase domain superfamily / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Butyrophilin subfamily 2 member A1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsRamesh, A. / Fuller, J.R. / Roy, S. / Adams, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Sci Rep / Year: 2025
Title: Mapping the extracellular molecular architecture of the pAg-signaling complex with α-Butyrophilin antibodies.
Authors: Amrita Ramesh / Sobhan Roy / Tomasz Slezak / James Fuller / Hortencia Graves / Murad R Mamedov / Alexander Marson / Anthony A Kossiakoff / Erin J Adams /
Abstract: Target cells trigger Vγ9Vδ2 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vγ9Vδ2 T cell ...Target cells trigger Vγ9Vδ2 T cell activation by signaling the intracellular accumulation of phospho-antigen metabolites (pAgs) through Butyrophilin (BTN)-3A1 and BTN2A1 to the Vγ9Vδ2 T cell receptor (TCR). An incomplete understanding of the molecular dynamics in this signaling complex hampers Vγ9Vδ2 T cell immunotherapeutic efficacy. A panel of engineered α-BTN3A1 and α-BTN2A1 antibody (mAb) reagents was used to probe the roles of BTN3A1 and BTN2A1 in pAg signaling. Modified α-BTN3A1 mAbs with increased inter-Fab distances establish that tight clustering of BTN3A1 is not necessary to stimulate Vγ9Vδ2 T cell activation, and that antagonism may occur through occlusion of a critical binding interaction between BTN3A1 and a yet unknown co-receptor. Finally, a panel of additional α-BTN2A1 antagonists utilize different biophysical mechanisms to compete with Vγ9Vδ2 TCRs for BTN2A1 binding. The complex structures of BTN2A1 ectodomain and Fabs from three antagonist mAbs provide molecular insights into BTN2A1 epitopes critical for pAg-signaling.
History
DepositionSep 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Butyrophilin subfamily 2 member A1
H: Human IgG1 Fragment Antibody Heavy Chain
L: Human IgG1 Fragment Antibody Light Chain
N: Human variable heavy-chain domain Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,7328
Polymers88,8474
Non-polymers8854
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Butyrophilin subfamily 2 member A1


Mass: 25741.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: BTN2A1 ectodomain with N- and C-terminal linkers. N-terminal (ADLQ) and C-terminal (VSPCGSGLEVLFQ) residues are disordered and not modeled. 3 glycans are denoted as NAG.
Source: (gene. exp.) Homo sapiens (human) / Gene: BTN2A1, BT2.1, BTF1 / Plasmid: pAc-GP67a / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q7KYR7
#2: Antibody Human IgG1 Fragment Antibody Heavy Chain


Mass: 24775.504 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Heavy chain of Herceptin Fab scaffold with CDR loops modified through phage-display evolution. (CDR1: FSSSI) (CDR2: SIYSSSGYTY) (CDR3: IEYGRGYWDAF). N-terminal (EIS) is a linker/restriction ...Details: Heavy chain of Herceptin Fab scaffold with CDR loops modified through phage-display evolution. (CDR1: FSSSI) (CDR2: SIYSSSGYTY) (CDR3: IEYGRGYWDAF). N-terminal (EIS) is a linker/restriction enzyme artifact and first residue of Fab, and disordered and not modeled.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Antibody Human IgG1 Fragment Antibody Light Chain


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Light chain of Herceptin Fab scaffold with CDR loops modified through phage-display evolution. (CDR1: VSSAV) (CDR2: IYSASSLY) (CDR3: SSSSLI). N-terminal (S) residue is a linker/restriction enzyme artifact.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: RH2.2 / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Antibody Human variable heavy-chain domain Nanobody


Mass: 15071.431 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human variable heavy-chain domain Nanobody specific for human Fab kappa light chain with a hexahistidine tag and linker (HHHHHHGENLYFQGS). Tag and linker are disordered and not shown.
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria)
#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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / 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
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
IDNameTypeEntity IDParent-IDSource
1Complex of Dimeric Human BTN2A1 ectodomain with TCR-Blocking 2A1.12 Fab and bulking NanobodyCOMPLEX#1-#40MULTIPLE SOURCES
2Human BTN2A1 ectodomainORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
3Anti-BTN2A1 Fab 2A1.12ORGANELLE OR CELLULAR COMPONENT#2-#31RECOMBINANT
4Heavy chain of 2A1.12 FabORGANELLE OR CELLULAR COMPONENT#23RECOMBINANT
5Light chain of 2A1.12 FabORGANELLE OR CELLULAR COMPONENT#33RECOMBINANT
6Variable heavy chain domain nanobodyORGANELLE OR CELLULAR COMPONENT#41RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
44
51NO
61NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
65Homo sapiens (human)9606
76Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Trichoplusia ni (cabbage looper)7111
43Escherichia coli BL21(DE3) (bacteria)469008
54Escherichia coli BL21(DE3) (bacteria)469008
65Escherichia coli BL21(DE3) (bacteria)469008
76Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
SpecimenConc.: 0.85 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5438
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2EPU3.5image acquisition
4CTFFIND4.1.14CTF correction
5RELION5.0-beta2CTF correction
8PHENIX1.21.1_5286model fitting
10RELION5.0-beta2initial Euler assignment
11RELION5.0-beta2final Euler assignment
13RELION5.0-beta23D reconstruction
14PHENIX1.21.1_5286model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 898426
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213773 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDSource nameType
18DFW

8dfw

A8DFWA1PDBexperimental model
2HAlphaFoldin silico model
3LAlphaFoldin silico model
4NAlphaFoldin silico model

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