Protein crystallization vapor diffusion experiments

Figure 6 Movement on the phase diagram in a vapor diffusion experiment. The simultaneous increase of both precipitant and protein concentrations corresponds to a diagonal movement in the phase diagram. Once crystal nucleation occurs, the growing crystals consume the protein in the solution, until the solution is no longer supersaturated.

Figure 12.19 The vapor diffusion experiment (hanging drop device shown) relies on water evaporating from the protein-containing droplet and diffusing away to the reservoir solution. Along with the protein, the droplet contains some crystallizing agent such as salt or PEG. The water evaporates until the droplet reaches chemical equilibrium with the reservoir solution. This evaporation simultaneously increases the protein and crystallization agent concentration that will, in the ideal case, cause nucleation and growth of a few protein crystals.

An important feature of protein crystal growth experiments is the need to carry out crystallization trials with very small quantities of scarce and expensive materials. When experiments are carried out in such small volumes (typically, 5—100 ju.1), it becomes difficult to define and control solution properties. The situation becomes particularly complicated when vapor diffusion or other nonequilibrium approaches to crystal growth are used, as these produce different and changing conditions throughout the small volumes involved. 

We therefore describe the basis of macromolecular crystallography and provide a summary of how to understand the results of a crystallographic experiment. We start with a mathematical description of what a crystal means in terms of symmetry this applies to all crystals, whether macromolecular or not. Later, we describe how protein crystals grow by using the hanging drop and sitting drop vapor diffusion methods this explains why protein crystals are so fragile and scatter X-rays very weakly. 

FIGURE 30.8 Principle of the method of vapor diffusion in hanging drops. A droplet (0.1-1 pU) of a solution of protein or protein complex is mixed with crystallization buffer (typically in a 1 1 ratio), suspended above a reservoir containing the crystallization buffer ( 0.1-0.5mL) and allowed to equilibrate. Because of the concentration gradient, water is transferred from the drop to the reservoir via the vapor phase. As a result, the concentrations of protein and precipitant in the drop increase. If a supersaturated state is reached, the protein will then precipitate. The precipitate is usually amorphous, but crystals will form in successful experiments. 

Crystallization experiments carried out on hen egg white lysozyme (HEWL) demonstrated that, under comparable or even lower supersaturation ratio, induction times for protein crystals grown on polypropylene membranes are lower than those reported in literature for conventional vapor diffusion techniques (Di Proflo et al. 

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