Picolitre-scale crystallization of membrane proteins

Crystallization of membrane proteins in lipidic mesophases by the standard in meso method is extremely efficient in that small amounts of valuable protein are required per trial. Here it is shown that it is possible to reduce the requisite amount of protein (and lipid) by two orders of magnitude into the picolitre volume range. Successful crystallizations have been performed with two integral membrane proteins, bacteriorhodopsin and the vitamin B₁₂ receptor, BtuB, using volumes of mesophase corresponding to 210 pl of protein solution (2-4 ng protein) and 320 pl of lipid. The total dead volume of the system is 1 µl. This means that thousands of crystallization trials can be performed with just micrograms of the target. Thus, for a given amount of protein, which is often in short supply, the likelihood of obtaining crystals is significantly enhanced. The reproducibility of crystallogenesis and of volume delivery at this picolitre scale is described. This advance will contribute to broadening the range of membrane proteins that yield to structure determination.