Phase Determination And Three Dimensional Model Building In Protein Crystallography

Protein crystallography plays an essential role in the development of structural biology. It includes protein sample preparation, crystal cultivation and optimization, data collection, phase determination and model building etc. Among all of these, the phase determination and model building process is very important for our research. With crystallographers’ great efforts on developing methods and softwares for data processing and structure determination, the automation level and capacity of crystallography have been improved significantly. In addition, the wide use of high quality light sources, such as synchrotron radiation and X-ray free electron laser(XFEL), and new types of detectors during data collection, have improved the quality of the diffraction data dramatically. As a result, solving a crystal structure becomes easier, and it promotes the development of structural biology. Now more and more researchers begin to clarify their scientific problems with the usage of protein crystallography.The barriers for structure determination of distinct protein crystals are always different. The development of crystallography has extended the limits for structure determination, and makes it possible to solve phases for the data with relatively low resolution and poor diffraction. However, we cannot always get the desired diffraction data, which is good enough for initial phase solving, in many cases, especially for the membrane proteins and large protein complexes. Meanwhile, the optimization of those crystals may take a lot of time and efforts. In this situation, the structure determination with the data available will be very important. This requires that the researchers should know crystallography well and gain much practical experience.In this paper, I will briefly introduce the principle of X-ray crystallography, and talk about the data collection and phase solving processes of several representative cases. The molecular replacement and anomalous scattering method will be discussed in detail. In order to make the whole thesis better for understanding, I will mainly focus on nucleic acid recognition proteins(TALE/DNA, PPR/RNA and Lsm/RNA), glucose transporters(XylE and GLUT1), γ-secretase related proteins(DpNCT and PSH), and some other typical examples. We’ve adopted different strategies for the final determination of the structures listed above. I hope this real case study will provide valuable information for the other researchers in structural biology.