From test tube to plate: a simple procedure for the rapid preparation of microcrystallization experiments using the cubic phase method

Abstract

Crystals of transmembrane proteins for X-ray diffraction experiments may be grown either by employing mixed protein–detergent complexes, or in a matrix of liquid-crystalline membraneous material forming a lipidic cubic phase (in cubo). Widespread use of thein cubomethod has been severely hampered by its tediousness and the large amounts of protein required. Here a simple procedure is presented that by virtue of its simplicity and small setup size substantially reduces the preparation time as well as the amount of protein. Crystallization trials are set up in conventional multi-well plates using a semi-automatic dispenser-driven microsyringe. The microprocedure is amenable to full automation and further miniaturization. Its feasibility is demonstrated by screening for new crystallization conditions for bacteriorhodopsin using volumes ofca200 nl of lipidic cubic phase. New crystallization conditions were identified that avoid the necessity of weighing solid precipitation agents.