Computational protocol: The Vα14 invariant natural killer T cell TCR forces microbial glycolipids and CD1d into a conserved binding mode

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Protocol publication

[…] Crystals of both mCD1d–glycolipid–TCR complexes were grown at 22.3°C by sitting drop vapor diffusion while mixing 0.5 µl of protein with 0.5 µl of precipitate (18% polyethylene glycol 3350 and 0.2 M ammonium citrate dibasic for GalA-GSL; 20% polyethylene glycol 3350 and 0.1 M citrate, pH 5, for BbGL-2c). Crystals were flash-cooled at 100 K in mother liquor containing 20% glycerol. Diffraction data were collected at the Stanford Synchrotron Radiation Laboratory beamline 7.1 and processed with the HKL2000 software suite (). The mCD1d–GalA-GSL–TCR crystal belongs to space group C2221 with cell parameters a = 79.0 Å, b = 191.2 Å, and c = 151.2 Å. The mCD1d–BbGL-2c–TCR crystal also belongs to space group C2221 with cell parameters a = 78.0 Å, b = 188.4 Å, and c = 149.8.2 Å.The asymmetric unit contains one mCD1d–glycolipid–TCR molecule with an estimated solvent content of 56.9% for GalA-GSL and 55.3% for BbGl-2c complex. The structures were determined by molecular replacement using MOLREP as part of the CCP4 suite (; ) using the protein coordinates from the mCD1d-iGB3 structure (Protein Data Bank ID 2Q7Y; ), followed by the variable domain of the mouse Vα14Vβ8.2 TCR () and, finally, the constant domain (from PDB ID 3HE6) as the search model. The molecular replacement solution with the complete TCR revealed that the constant domain was slightly tilted in both of our structures. After the molecular replacement solution was obtained containing both mCD1d and TCR, the model was rebuilt into σA-weighted 2Fo-Fc and Fo-Fc difference electron density maps using the program COOT (). The final refinement steps were performed using the TLS (translation, libration, and screw axis) procedure in REFMAC () with five anisotropic domains (α1-α2 domain of CD1d, including carbohydrates and glycolipid, α3 domain, β2M, variable domain, and constant domain of TCR). The mCD1d–GalA-GSL–TCR structure was refined to 2.74 Å with a final Rcryst and Rfree of 19.8% and 25.3%, respectively. The mCD1d–BbGL-2c–TCR structure was refined to 2.8 Å with a final Rcryst and Rfree of 20.3% and 25.9%, respectively. The quality of the model was examined with the program Molprobity (). […]

Pipeline specifications

Software tools Molrep, CCP4, Coot, MolProbity
Application Protein structure analysis
Organisms Borreliella burgdorferi