Appropriate crystal growth rate is a critical factor in obtaining a high-quality crystal. Traditionally, optimizing crystal growth rate is usually through increasing or decreasing protein and precipitant concentration. Here, a method will be shown to control nucleation rate and increase crystal size. By adding additional water to crystal drop we can decrease protein and precipitant concentration at an equal proportion, which allows a lower supersaturate environment at the initial stage of crystallization. For this reason, over-nucleated protein molecules can only bind to the surface of a limited number of growing centers. Then, the additional water will leave a crystal drop in the reservoir solution after equilibration and does not change the final ratio of protein and precipitant reagent. As a result, the total crystal number decreases but the crystal size has a dramatic increase, as well as the morphology of crystals. In the current study, pleckstrin homology (PH) domain of 3-phosphoinositide-dependent protein kinase 1 protein (PDK1) was taken as an example to illustrate this phenomenon and its underlying mechanism, which aims to provide a strategy for crystal optimization.

Keywordsnucleation rate, crystal size, water, PDK1