Protein crystallization

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Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017

Vapor diffusion is the most commonly used method for protein crystallization. Two primary setups for vapor diffusion exist: sitting drop and hanging drop (Figure 6.2). Each method has its advantages and drawbacks; a comparison of the two methods is given in Table 6.1. Both make use of a sealed chamber to allow solvent exchange between two solutions: drop and reservoir. The drop contains protein, while the reservoir does not. The reservoir solution (100–1000 μL or less), also referred to as the crystallization solution, usually contains three constituents: buffer, precipitant, and salt. (The following section describes in detail what these individual parameters are thought to do.) The drop (1–10 μL or less) is a mixture of the reservoir solution and protein sample, also called mother liquor. The ratio of this mixture can vary but is often 1:1. Upon creating a sealed environment, volatile constituents in the drop, usually water, will slowly leave the drop to travel to the reservoir solution. This process continues until the concentrations of the precipitant, salt, and so on in the drop solution approach their respective concentrations in the reservoir solution. Slowly, the concentrations of all nonvolatile components within the drop effectively increase.

Influence of rapid cooling on crystal nucleation in lysozyme crystallization solutions of low supersaturation

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Published in Phase Transitions, 2021

Petya P. Elenska, Ivaylo L. Dimitrov

Protein crystallization represents a set of processes that lead to the formation of ordered solid protein phase. Generally, it requires protein-enriched water-based medium which also contains specific low or high molecular mass substances (precipitants) that are crucial for the inception of the overall process. Designing reliable systems for protein crystal formation with such a complex medium often is more an art than a science [1], especially if one wants to obtain crystals of previously non-crystallized protein molecules. This phenomenon of self-assembly could take days, weeks or even months, and manifest the bottleneck in the molecular structure determination via X-ray or neutron diffraction techniques. Still, the problem of obtaining a crystal phase might not be the only one. Of special importance is the obtaining of crystalline material suitable for further processing. That issue is mainly dependent on the stochastic nature of crystal nucleation – the process of the very generation of a new phase. Hence it will always persist in some form even for largely investigated protein crystallization systems such as those involving the protein lysozyme. Most of the academic studies focus on elucidating the way in which protein crystals emerge in the mother liquor as well as their subsequent growth [2,3]. Both crystal nucleation and growth determine the final crystallization outcome and the accumulated knowledge helps in understanding the mechanisms through which the crystals assemble. A variety of physical or chemical factors may influence protein crystal nucleation and growth. The formation of protein crystals could strongly respond to changes in pH of the solution, temperature, magnetic or electric fields, type of the precipitant, protein or precipitant concentration, solution purity, buffer composition, and so forth [4–6]. Even some features of the experimental approach could modify the crystallization at all other conditions being constant. Moreover, the desired crystallization could happen in a narrow range of conditions [7,8]. This means that an inconsistent mix of factors, or their strength in particular, may result in the formation of non-crystalline phase or unsatisfactory crystallization outcome. Such a complex prerequisite for a diversity in the crystallization behavior of proteins continues to sustain protein crystal nucleation and growth as a fascinating area for investigations.