A guide to membrane protein X‐ray crystallography

Membrane proteins play critical physiological roles in all organisms, from ion transport and signal transduction to multidrug resistance. Elucidating their 3D structures is essential for understanding their functions, and this information can also be exploited for structure‐aided drug discovery efforts. In this regard, X‐ray crystallography has been the most widely used technique for determining the high‐resolution 3D structures of membrane proteins. However, the success of this technique is dependent on efficient protein extraction, solubilization, stabilization, and generating diffracting crystals. Each of these steps can impose great challenges for membrane protein crystallographers. In this review, the process of generating membrane protein crystals from protein extraction and solubilization to structure determination is discussed. In addition, the current methods for precrystallization screening and a few strategies to increase the chance of crystallizing challenging membrane proteins are introduced. X‐ray crystallography is a powerful technique for determining the 3D structure of membrane proteins; however, practitioners face a variety of unique challenges, including expression difficulties, the instability of membrane proteins upon detergent extraction, and a lower probability of crystallization. This review provides a guide to membrane protein X‐ray crystallography with an eye to the difficulties faced and covers novel techniques to overcome them, including crystallization chaperones and in meso crystallography.