research papers
In a classical vapor diffusion crystallization, the protein solution is mixed in a 1:1 ratio with the reservoir solution, containing one or more precipitant species, after which the two are placed in an enclosed chamber. As the vapor pressure is lower for the reservoir solution, due to its higher solute concentration, there is a net transfer of water through the vapor phase from the protein droplet to the reservoir. In theory, the initial conditions in the droplet are such that the protein is in either a metastable or undersaturated state with respect to crystal nucleation. The loss of water serves to both concentrate the protein and the precipitant concentrations within the drop, bringing the protein past the metastable point to nucleation. The equilibration rate is a function of the precipitant(s) used, their concentration, the temperature, the distance between the two surfaces, and the droplet to reservoir volume ratio. For a given reservoir volume smaller droplets equilibrate faster, the rate being inversely linear with the droplet volume. In attempts to maximize the number of crystallization trials, and as crystals in the 100 – 200 μm size range are sufficient, it has currently become standard practice to use starting droplet volumes of 2 - 4 μl, with reservoir volumes typically in the 200 to 500 μl range. The equilibration rates are maximized, and for most common salt concentrations and higher concentrations of polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) one can reasonably estimate that equilibration has occurred within 3 to 6 days at room temperature. Crystals appearing after this time are essentially grown under batch conditions. We experimentally find that altering the reservoir to droplet volume ratio, by changing the reservoir volume, from 50:1 (high ratio) to 5:1 (low ratio), on average increases the equilibration time by approximately 50 % when tested with solutions of 50% MPD, 1.5 M NaCl, or 30 % PEG400. However, experiments with two proteins, chicken egg white lysozyme and concanavalin a, showed an unexpected trend of slightly faster nucleation and larger crystals in the lowest ratio experiments.