Begin by preparing a 10%bicelle stock solution containing the lipids and detergent. To do so, take the pre-dried lipid, which is a mix of five mole percent cholesterol and 95 mole percent DMPC, and add the CHAPSO detergent solution in deionized water to it. Using a water bath sonicater, resuspend the lipid detergent mixture by sonicating it in ice-chilled water under continuous power until the solution is clear.
Then with the aid of a 0.2 micron centrifugal filter, remove any undissolved components. Next, working on the ice, combine the resultant bicelle stock solution with the pre-prepared protein sample at a one to four volumetric ratio. Incubate the protein bicelle mixture on ice for 30 minutes.
For crystallization, in a 48 well plate mix 0.5 or one microliter of the protein bicelle mixture with an equal volume of crystallization reservoir solution. Then set up crystallization in a hanging drop vapor diffusion mode using the 48 well plate and incubate the crystallization setup at 20 degrees Celsius. On the following day, check the crystallization trays to ensure the cover glass is sealed properly.
Check on the crystal growth at least once daily using a tabletop stereo microscope equipped with a polarizer. Lastly, soak the protein crystals in 0.2 molar sodium malate and flash freeze them in 50 or 100 micrometer cryo loops using liquid nitrogen. Mature crystals that were suitable for data collection usually appeared within one to two weeks and generally had the dimensions of 50 microns by 100 microns by two microns.
