Current pivotal strategies leading a difficult target protein to a sample suitable for crystallographic analysis

Crystallographic structural analysis is an essential method for the determination of protein structure. However, crystallization of a protein of interest is the most difficult process in the analysis. The process is often hampered during the sample preparation, including expression and purification. Even after a sample has been purified, not all candidate proteins crystallize. In this mini-review, the current methodologies used to overcome obstacles encountered during protein crystallization are sorted. Specifically, the strategy for an effective crystallization is compared with a pipeline where various expression hosts and constructs, purification and crystallization conditions, and crystallization chaperones as target-specific binder proteins are assessed by a precrystallization screening. These methodologies are also developed continuously to improve the process. The described methods are useful for sample preparation in crystallographic analysis and other structure determination techniques, such as cryo-electron microscopy.