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- EMDB-47104: CryoEM Structure of Human BTN2A1 ectodomain in complex with TCR-b... -

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Basic information

Entry
Database: EMDB / ID: EMD-47104
TitleCryoEM Structure of Human BTN2A1 ectodomain in complex with TCR-blocking 2A1.12 Fab
Map dataRELION multi-body refinement, body 1 full map
Sample
  • Complex: Complex of Dimeric Human BTN2A1 ectodomain with TCR-Blocking 2A1.12 Fab and bulking Nanobody
    • Organelle or cellular component: Human BTN2A1 ectodomain
      • Protein or peptide: Butyrophilin subfamily 2 member A1
    • Organelle or cellular component: Anti-BTN2A1 Fab 2A1.12
      • Organelle or cellular component: Heavy chain of 2A1.12 Fab
        • Protein or peptide: Human IgG1 Fragment Antibody Heavy Chain
      • Organelle or cellular component: Light chain of 2A1.12 Fab
        • Protein or peptide: Human IgG1 Fragment Antibody Light Chain
    • Organelle or cellular component: Variable heavy chain domain nanobody
      • Protein or peptide: Human variable heavy-chain domain Nanobody
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsComplex / Antibody / Immune Recognition / SIGNALING PROTEIN / IMMUNE SYSTEM
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsRamesh A / Fuller JR / Roy S / Adams E
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47104.png

Downloads & links

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Map

FileDownload / File: emd_47104.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION multi-body refinement, body 1 full map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 384 pix.
= 408.96 Å		1.07 Å/pix.
x 384 pix.
= 408.96 Å		1.07 Å/pix.
x 384 pix.
= 408.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.005612341 - 0.020239888
Average (Standard dev.)-0.000012277883 (±0.00040133198)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 408.96002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47104_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION multi-body refinement, body 1 half map 2

Fileemd_47104_half_map_1.map
AnnotationRELION multi-body refinement, body 1 half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION multi-body refinement, body 1 half map 1

Fileemd_47104_half_map_2.map
AnnotationRELION multi-body refinement, body 1 half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of Dimeric Human BTN2A1 ectodomain with TCR-Blocking 2A1....

EntireName: Complex of Dimeric Human BTN2A1 ectodomain with TCR-Blocking 2A1.12 Fab and bulking Nanobody
Components
  • Complex: Complex of Dimeric Human BTN2A1 ectodomain with TCR-Blocking 2A1.12 Fab and bulking Nanobody
    • Organelle or cellular component: Human BTN2A1 ectodomain
      • Protein or peptide: Butyrophilin subfamily 2 member A1
    • Organelle or cellular component: Anti-BTN2A1 Fab 2A1.12
      • Organelle or cellular component: Heavy chain of 2A1.12 Fab
        • Protein or peptide: Human IgG1 Fragment Antibody Heavy Chain
      • Organelle or cellular component: Light chain of 2A1.12 Fab
        • Protein or peptide: Human IgG1 Fragment Antibody Light Chain
    • Organelle or cellular component: Variable heavy chain domain nanobody
      • Protein or peptide: Human variable heavy-chain domain Nanobody
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Complex of Dimeric Human BTN2A1 ectodomain with TCR-Blocking 2A1....

SupramoleculeName: Complex of Dimeric Human BTN2A1 ectodomain with TCR-Blocking 2A1.12 Fab and bulking Nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Human BTN2A1 ectodomain

SupramoleculeName: Human BTN2A1 ectodomain / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Anti-BTN2A1 Fab 2A1.12

SupramoleculeName: Anti-BTN2A1 Fab 2A1.12 / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Heavy chain of 2A1.12 Fab

SupramoleculeName: Heavy chain of 2A1.12 Fab / type: organelle_or_cellular_component / ID: 4 / Parent: 3 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: Light chain of 2A1.12 Fab

SupramoleculeName: Light chain of 2A1.12 Fab / type: organelle_or_cellular_component / ID: 5 / Parent: 3 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #6: Variable heavy chain domain nanobody

SupramoleculeName: Variable heavy chain domain nanobody / type: organelle_or_cellular_component / ID: 6 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Butyrophilin subfamily 2 member A1

MacromoleculeName: Butyrophilin subfamily 2 member A1 / type: protein_or_peptide / ID: 1
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.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.741371 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: ADLQFIVVGP TDPILATVGE NTTLRCHLSP EKNAEDMEVR WFRSQFSPAV FVYKGGRERT EEQMEEYRGR TTFVSKDISR GSVALVIHN ITAQENGTYR CYFQEGRSYD EAILHLVVAG LGSKPLISMR GHEDGGIRLE CISRGWYPKP LTVWRDPYGG V APALKEVS ...String:
ADLQFIVVGP TDPILATVGE NTTLRCHLSP EKNAEDMEVR WFRSQFSPAV FVYKGGRERT EEQMEEYRGR TTFVSKDISR GSVALVIHN ITAQENGTYR CYFQEGRSYD EAILHLVVAG LGSKPLISMR GHEDGGIRLE CISRGWYPKP LTVWRDPYGG V APALKEVS MPDADGLFMV TTAVIIRDKS VRNMSCSINN TLLGQKKESV IFIPESFMPS VSPCGSGLEV LFQ

UniProtKB: Butyrophilin subfamily 2 member A1

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Macromolecule #2: Human IgG1 Fragment Antibody Heavy Chain

MacromoleculeName: Human IgG1 Fragment Antibody Heavy Chain / type: protein_or_peptide / ID: 2
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.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.775504 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSSSSIHWV RQAPGKGLEW VASIYSSSGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARIEYGRGYW DAFDYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP E PVTVSWNS ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSSSSIHWV RQAPGKGLEW VASIYSSSGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARIEYGRGYW DAFDYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP E PVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHT

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Macromolecule #3: Human IgG1 Fragment Antibody Light Chain

MacromoleculeName: Human IgG1 Fragment Antibody Light Chain / type: protein_or_peptide / ID: 3
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.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: Human variable heavy-chain domain Nanobody

MacromoleculeName: Human variable heavy-chain domain Nanobody / type: protein_or_peptide / ID: 4
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.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.071431 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
HHHHHHGENL YFQGSQVQLQ ESGGGLVQPG GSLRLSCAAS GRTISRYAMS WFRQAPGKER EFVAVARRSG DGAFYADSVQ GRFTVSRDD AKNTVYLQMN SLKPEDTAVY YCAIDSDTFY SGSYDYWGQG TQVTVSS

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.85 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 5438 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.9 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 898426
Startup modelType of model: OTHER / Details: RELION 3D initial model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0-beta2) / Number images used: 213773
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0-beta2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0-beta2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

8dfw

chain_id: A, source_name: PDB, initial_model_type: experimental model
chain_id: H, source_name: AlphaFold, initial_model_type: in silico model
chain_id: L, source_name: AlphaFold, initial_model_type: in silico model
chain_id: N, source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation
Output model

PDB-9dpe:
CryoEM Structure of Human BTN2A1 ectodomain in complex with TCR-blocking 2A1.12 Fab

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