Vapor diffusion is still the most commonly employed technique used to grow protein crystals for X-ray diffraction. In this approach, a protein solution is mixed with a reservoir solution, and small-volume droplets of this mixture are equilibrated against the higher-volume reservoir solution. The droplet solution, having a lower solute concentration, has a higher vapor pressure in comparison to the reservoir solution, which causes water to diffuse over time from the droplet to the reservoir solution until the two are at equilibrium. Water evaporation from the droplet causes an increase in protein and precipitant concentrations, which can bring the protein past the metastable phase to the crystal nucleation phase.

In this application note, we assess the effects of humidity on the droplet size and composition during the course of crystallization plate preparation.