Purified CorC TM domain proteins were mixed with MgCl2 at a final concentration of 50 mM, incubated for 30 min, and then mixed with monoolein (Nu-Chek, USA) at a ratio of 2:3 (w/w) with a coupled syringe mixer to generate LCP. The robotic LCP crystallization trial was performed by dispensing 50 nl of LCP drops onto 96-well sandwich plates and overlaid with 700 nl of reservoir solution using a Gryphon LCP crystallization robot (Art Robbins Instruments, USA). All LCP crystals were obtained in a solution containing 10 mM ZnCl2, 100 mM sodium acetate (pH 4.0), and 40% polyethylene glycol 200 (PEG 200) at 18°C. Crystals typically appeared in 3 days and grew to their maximum size within 1 week. Crystals were then harvested in reservoir solution supplemented with 40% PEG 200 and 50 mM MgCl2 and flash-frozen with liquid nitrogen for x-ray diffraction experiments.

For crystallization by the vapor diffusion method, 1 μl of the TpCorC CBS domain proteins (residues 183 to 361, Y255A, and residues 202 to 361) was mixed with 1 μl of reservoir solution [0.4 M ammonium thiocyanate, 0.1 M sodium acetate (pH 4.5), and 15% PEG 4000 and 0.1 M CaCl2, 0.1 M Hepes (pH 7.5), and 5% PEG 8000] and stored at 18°C. Before flash freezing in liquid nitrogen, crystals were harvested with the corresponding cryoprotectant solutions: for the crystals of the TpCorC CBS domain protein (residues 183 to 361, Y255A), 30% glycerol, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate (pH 4.5), and 15% PEG 4000 and for the crystals of the TpCorC CBS domain protein (residues 202 to 361), 30% glycerol, 0.1 M CaCl2, 0.1 M Hepes (pH 7.5), and 5% PEG 8000.

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