High-throughput screening of a wide range of different conditions is typically required to obtain X-ray quality crystals of proteins for structure-function studies. The outcomes of individual experiments, i.e. the formation of gels, precipitates, microcrystals, or crystals, guide the search for and optimization of conditions resulting in X-ray diffraction quality crystals. Unfortunately, the protein will remain soluble in a large fraction of the experiments. In this paper, an evaporation-based crystallization platform is reported in which droplets containing protein and precipitant are gradually concentrated through evaporation of solvent until the solvent is completely evaporated. A phase transition is thus ensured for each individual crystallization compartment; hence the number of experiments and the amount of precious protein needed to identify suitable crystallization conditions is reduced. The evaporation-based method also allows for rapid screening of different rates of supersaturation, a parameter known to be important for optimization of crystal growth and quality. The successful implementation of this evaporation-based crystallization platform for identification and especially optimization of crystallization conditions is demonstrated using the model proteins of lysozyme and thaumatin.

Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin.

The quality, size, and number of protein crystals grown under conditions of continuous solvent extraction are dependent on the rate of solvent extraction and the initial protein and salt concentration. An increase in the rate of solvent extraction leads to a larger number of crystals. The number of crystals decreases, however, when the experiment is started with an initial protein concentration that is closer to the solubility boundary. Here we develop a kinetic model capable of predicting changes in the number and size of protein crystals as a function of time under continuous evaporation. Moreover, this model successfully predicts the initial condition of drops that will result in gel formation. We test this model with experimental crystal growth data of hen egg white lysozyme for which crystal nucleation and growth rate parameters are known from other studies. The predicted and observed rates of crystal growth are in excellent agreement, which suggests that kinetic constants for nucleation and crystal growth for different proteins can be extracted by applying a kinetic model in combination with observations from a few evaporation-based crystallization experiments.