High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

Environments with varying temperatures have been shown to beneficially increase the probability of obtaining protein crystals. Therefore, a cycling temperature strategy (CTS) has been proposed for protein crystallization screening. During the practical application of this strategy, it is necessary to know the effective temperature range that promotes crystallization to design a suitable temperature program. In this paper, the effects of different temperature ranges on lysozyme crystallization (or more specifically, nucleation) were investigated. The results show that a small periodic variation in the temperature range of as little as 0.4 K can have a significant effect on the crystallization success rate under some crystallization concentration conditions, confirming that crystallization of lysozyme is very sensitive to temperature variation. Because practical protein crystallization is always performed in an environment with slight temperature variations, the sensitivity of protein crystallization to temperature may provide an explanation for the poor reproducibility of protein crystallization. Further investigation of the CTS on lysozyme crystallization showed that a cycling temperature strategy exerts an effect on protein crystallization by altering the supersaturation caused by changes in temperature.

Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.

The contact angle is an important parameter that is essential for studying interfacial phenomena. The contact angle can be measured using commercially available instruments. However, these well-developed instruments may not function or may be unsuitable for use in some special environments. A simulated microgravity generated by a large gradient magnetic field is such an environment in which the current measurement instruments cannot be installed. To measure the contact angle in this environment, new tools must be designed and manufactured to be compatible with the size and physical environment. In this study, we report the development and construction of a new setup that was specifically designed for use in a strong magnetic field to measure the contact angle between a levitated droplet and a solid surface. The application of the setup in a large gradient magnetic field was tested, and the contact angles were readily measured.

Most protein crystallisation begins from heterogeneous nucleation; in practice, crystallisation typically occurs in the presence of a solid surface in the solution. The solid surface provides a nucleation site such that the energy barrier for nucleation is lower on the surface than in the bulk solution. Different types of solid surfaces exhibit different surface energies, and the nucleation barriers depend on the characteristics of the solid surfaces. Therefore, treatment of the solid surface may alter the surface properties to increase the chance to obtain protein crystals. In this paper, we propose a method to modify the glass cover slip using a self-assembled monolayer (SAM) of functional groups (methyl, sulfydryl and amino), and we investigated the effect of each SAM on protein crystallisation. The results indicated that both crystallisation success rate in a reproducibility study, and crystallisation hits in a crystallisation screening study, were increased using the SAMs, among which, the methyl-modified SAM demonstrated the most significant improvement. These results illustrated that directly modifying the crystallisation plates or glass cover slips to create surfaces that favour heterogeneous nucleation can be potentially useful in practical protein crystallisation, and the utilisation of a SAM containing a functional group can be considered a promising technique for the treatment of the surfaces that will directly contact the crystallisation solution.