Lipidic Cubic Mesophase Protein Crystallization

Crystallization trials began with the reconstitution of the protein into the bilayer of the lipidic cubic mesophase following a standard protocol^{32 (link)}. The protein solution at 12 mg/mL was homogenized with 7.9 MAG in equal parts by volume using a coupled syringe device at room temperature (20–22 °C). Approximately 20 μL of protein-laden mesophase was transferred into a 0.5 mL Hamilton syringe containing 0.4 mL precipitant solution (0.2 % (v/v) MPD, 0.1 M sodium chloride, 0.1 M sodium citrate pH 5.6) using a narrow bore coupler^{32 (link)}, as described above for GPCR crystallization. The syringe was incubated for 21 d at 20 °C for crystal growth. After separating the bathing solution from the crystal-laden LCP, excess precipitant was absorbed by mixing in 3–5 μL molten 7.9 MAG. This procedure produced optically clear LCP in which microcrystals were dispersed ready for loading into the reservoir of the LCP injector, as described above.