A simple and efficient innovation of the vapor-diffusion method for controlling nucleation and growth of large protein crystals

The rate of water vaporization in the vapor-diffusion method is critical for the protein crystallization process. Present methods, however, allow little or no control of the equilibration rates. This paper presents a relatively simple innovation of the conventional vapor-diffusion method by introducing a capillary barrier (for hanging drop) or a punched film barrier (for both hanging and sitting drop) between drop and reservoir, which can be beneficial in controlling the water vaporization rate, thereby promoting growth of large protein crystals. The crystallization experiments for lysozyme, trichosanthin and a novel neurotoxin BmK Mu9 show that this modified vapor-controlling-diffusion method is very effective for producing large protein crystals. The improved technique can be routinely used as a method for the preparation of other macromolecular and small-molecule crystals whose crystallization involves vaporization of water.