**Abstract**

Nowadays, X-ray crystallography is one of the most popular structural biology methods. Successful crystallization depends not only on the quality of the protein sample, precipitant composition, pH or other biophysical and biochemical parameters, but also largely on the use of crystallization technique. Some proteins are difficult to be crystallized using basic crystallization methods; therefore, several advanced methods for macromolecular crystallization have been developed. This chapter briefly reviews the most promising advanced crystallization techniques and strategies as one of the efficient tools for crystallization of macromolecules. Crystallization in capillaries, gels, microfluidic chips, electric and magnetic fields as well as crystallization under microgravity condition and crystallization in living cells are briefly described.

**Keywords:** protein crystallization, protein crystal, advanced methods, crystallization strategies, biocrystallogenesis

### **1. Introduction**

Macromolecular crystallization was invented accidentally in the late 19th century. The initial goal of crystallization processes was to purify and to prove the chemical purity of the examined chemical compound. One of the first achievements in macromolecular crystallization were crystals of hen-egg albumin at the end of the 19th century and crystals of insulin in the 1920s [1, 2]. Since then, macromolecular crystallization has developed into a powerful tool for the threedimensional structure determination of nucleic acids, proteins as well as for larger macromolecular complexes.

A good-quality macromolecular crystal is needed in X-ray crystallography for obtaining a 3D model of corresponding nucleic acids, proteins, macromolecular complexes or larger biological assemblies, for instance, viruses or ribosomes. Nevertheless, macromolecular crystallization is a highly unpredictable process, which relies on a large number of chemical and biochemical parameters, such as the purity and concentration of the protein, type of precipitant, pH of the buffer, as well as on physical parameters, as temperature, time, pressure or vibrations, etc.

In order to crystallize the protein from the solution, the supersaturated state has to be reached using various crystallization techniques. Crystallization process can be divided into three stages - nucleation, crystal growth and termination. So-called phase diagrams typically illustrate these steps. For the investigation of the best conditions for crystal growth of the individual macromolecule, two general strategies are usually applied. Firstly, initial crystallization screening using commercially available screening kits is performed to explore the suitable conditions for crystal growth. Secondly, conditions yielding macromolecular crystals are systematically modified to allow the growth of the best-quality crystals adequate for X-ray diffraction analysis. Basic crystallization techniques, namely batch, vapour diffusion and free-interface diffusion are used for initial crystallization screening generally [3]. In addition, these techniques can be further modified to obtain as good-quality crystals as possible during the optimization of protein crystallization. However, some proteins cannot be easily crystallized and produced crystals in diffraction quality and thus initial hits need to be further improved. It is important to note that different crystallization methods screen the phase diagram from different points of view and hence affect the properties of the resulting crystals. Therefore, advanced biocrystallogenesis methods are recommended to be applied for screening or optimization of crystallization conditions.

This chapter reviews different advanced methods and strategies as efficient tools for crystallization of macromolecules. During the past few years, the field of protein crystallography has significantly developed regarding instrumentation. Different technologies require different sizes of crystals, for instance homogeneous nanocrystals for XFEL or big size crystals for neutron diffraction [4, 5]. This also drives the necessity to develop new and convenient methods for growing suitable high-quality protein crystals. For this purpose advanced crystallization methods and strategies have been developed.
