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Obtaining good quality protein crystals is still desirable for high resolution structural determination of the proteins using crystallography. The crystal quality is affected by the growth environment and growth methods. As frequently used methods in practical protein crystallization, sitting and hanging-drop methods are seldom compared in terms of crystal quality in the literatures. We performed a systematic comparison on the quality of the protein crystals grown in sitting and hanging-drop methods using different crystallization plates: cross-diffusion microbatch plate (CDM SD: CDM plate, sitting drop method; CDM HD: CDM plate, hanging drop method), traditional sitting and hanging-drop vapor diffusion plates (T SD: traditional plate, sitting drop method; and T HD: traditional plate, hanging drop method). Five different proteins, proteinase K (prk), lysozyme (lys), concanavalin A VI (con), catalase (cat) and α-Chymotrypsinogen A II (chy), were used. The crystal quality was compared in terms of the resolution limit, mosaicity and Wilson plot B factor. It was found that the crystals grown in CDM plate using the hanging drop method (CDM HD) exhibited the best morphology and the best crystal quality. X-ray diffraction tests showed that the CDM plate using hanging drop method is indeed a practical and useful method for obtaining high-quality protein crystals.

We have proposed a rational strategy for selecting a suitable pH of protein solution based on protein biochemical properties. However, it is difficult to use this strategy for biochemical properties unknown proteins. In this paper, a simpler and faster pH buffer strategy was proposed. An additional pH-controlling buffer was added to crystallization droplet mixed with protein solution and commercial crystallization reagents to adjust its pH. The results revealed that protein crystallization success rates were enhanced by this strategy due to expansion of the pH screening space, which was closely related with protein solubility. Thus, the possibility of reaching supersaturation was increased by using this strategy.

One well-known prerequisite for successful crystallization from solution is a supersaturated solution. To achieve supersaturation, many methods are known, among which solvent evaporation is a common approach. For protein crystallization, the most widely used method is vapor diffusion, in which solvent evaporation from the crystallization solution is the major reason for achieving supersaturation. The solvent evaporation rate may affect the actual concentration distribution in the crystallization solution, thereby influencing the crystallization process. To explore the effect of evaporation rate on protein crystallization, we used lysozyme as a model protein and studied the crystallization success rate at different evaporation conditions. Successful crystallization occurred only when both supersaturation and evaporation rates were in suitable ranges. This study demonstrates that both supersaturation level and the rate of reaching supersaturation (or solvent evaporation rate) are important for lysozyme crystallization. To increase the chance of obtaining crystals, manipulation of solvent evaporation rate is one choice. According to this assumption, we performed crystallization screening trials at different evaporation rates using three model proteins. The trials demonstrate that control of the evaporation rate during crystallization may provide more opportunities to obtain crystals.

Magnetic field has been found to be able to improve the quality of some protein crystals; containerless condition is considered to be beneficial for growing good-quality crystals. Nevertheless, there has been no report on combination of these two conditions for protein crystallization. In this paper, crystallization and dissolution of protein crystals in a levitated containerless droplet in a strong magnetic field has been successfully achieved. New phenomena were observed for both crystallization and dissolution processes.

As one of the best candidates for simulating the microgravity conditions in space, low gravity environments provided by applying an upward magnetic force have been considered to grow protein crystals. Since 2002, the stable and long-time durable microgravity generated by a superconducting magnet has been available for protein crystal growth. In this paper, for the first time, we grew protein crystals (orthorhombic lysozyme crystals) at quasi-microgravity. The present study showed that quasi-microgravity improves the crystal quality effectively and reproducibly. The application of strong magnetic field also improves the crystal quality. Furthermore, it is possible to know if microgravity is effective for the improvement of crystal quality or not by growing crystals in microgravity and hypergravity inside the superconducting magnet and comparing the crystal quality with each other. Such test will be useful to select promising proteins prior to the space experiments.

Chlorides with different paramagnetic cations such as Ni 2+ , Co 2+ and Mn 2+ were used as crystallizing agents instead of NaCl to crystallize hen egg-white lysozyme. NiCl 2 was found to give two types of crystals with different morphologies: one (roof-like) is a new type of orthorhombic P2 1 2 1 2 1 crystal with lattice constants a =79.0 A, b =80.8 A, and c =37.5 A; the second is an ordinary tetragonal crystal of its characteristic shape with a = b =80 A and c =38 A. The appearance of the roof-like shape became dominant in the presence of a magnetic field. In the case of using CoCl 2 and MnCl 2 , ordinary tetragonal crystals were formed. A striking fact was that the a -axis of the crystals oriented along the magnetic field when CoCl 2 was used, as opposed to the usual c -axis orientation. Large and optically perfect lysozyme crystals can be obtained in a magnetic field when NiCl 2 or MnCl 2 is used as a crystallizing agent. These profound effects of the paramagnetic cations may be caused by the coordination of Ni 2+ and Co 2+ ions to a lysozyme molecule, which was found by X-ray crystallography.