Protein crystallization

Crystallization was performed by the hanging drop vapor diffusion method. WT TCR I.29 and 8F10 proteins were concentrated to 5 mg/ml. I.29 crystals were grown from 20% 2-methyl-2,4-pentanediol, 100 mM sodium cacodylate at pH 6.0, and 50 mM calcium acetate. 8F10 crystals were grown in 14% polyethylene glycol (PEG) 3350 and 100 mM sodium acetate at pH 6.0. Both I.29 and 8F10 crystals were cryoprotected by a well solution plus 25% glycerol. To obtain the I.29/IA^g7-8E9E6ss complex crystals, IA^g7-8E9E6ss and I.29(β56A) mutant TCR were mixed at a molar ratio of 1:1 at the concentration of 10 mg/ml. The crystals were grown in 12% PEG 20000, 100 mM bicine (pH 9.0), and 20% ethylene glycol and then cryoprotected by the well solution plus 15% ethylene glycol. Similarly, IA^g7-8G9E/8F10 complex crystals were obtained by mixing IA^g7-8G9E and 8F10(α169A) mutant TCR at a 1:1 molar ratio at the concentration of 10 mg/ml. Crystals were grown in 11% PEG 3350 and 100 mM formate (pH 7.0) at room temperature and then cryoprotected by the well solution plus 30% glycerol. Human T1D3/DQ8-8E9E11ss complex was crystallized in 10% PEG 8000, 100 mM cacodylate (pH 6.2), and 400 mM NaCl at 4°C by mixing TCR T1D3 and DQ8-8E9E11ss at a 1:1 molar ratio at the concentration of 12 mg/ml and then cryoprotected by the well solution plus 30% glycerol.