Cryogenic protein crystallography is now the indispensable method in structural biology. In this method, protein crystals are rapidly cooled by low temperature nitrogen gas or liquid ethane, and diffraction intensity data are collected at cryogenic temperature. By applying this method, the X-radiation damage of protein crystals are drastically decreased, and we can easily obtain diffraction data even for highly radiation-sensitive protein crystals. This method has been applied to investigate the hydration structures around protein molecules and to analyze the structure of reaction-intermediates of proteins. Here, we briefly report the procedures and the experimental techniques using the newly developed devices and discuss the advantages and the disadvantages of this method. In addition, we describe the application of this method to crystallographically investigate the mechanism of the glassy transition observed in the vibrational states of protein molecules around 200 K.
