The second stage of the crystallization process, optimization, refines the chemical and physical parameters identified during screening to produce crystals of sufficient quality for X-ray diffraction analysis.  Optimization builds directly upon the success of the screening experiments.  The first tier optimization method takes the initial crystallization condition and varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner by changing both the drop volume ratio of protein to cocktail and the temperature of incubation (DVR/T).  The DVR/T method is proven for soluble proteins and ideally suited to manipulate the phase behavior of a protein:detergent complex used in membrane protein crystallization.

The DVR/T method maximizes efficiency by eliminating reformulation of the cocktail solutions for optimization.  In addition, by incorporating the same crystallization protocol for both screening and optimization trials the process eliminates complications that arise when converting crystallization conditions from one method to another.  The sample requirements for this technique are minimal; 25 mL of protein solution are required to set up 64 experiments.  These experiments sample a full matrix of 16 different drop chemistries at 4 unique temperatures.

Optimization and screening protocols use exactly the same microbatch-under-oil crystallization method (identical mineral oil, 1536 well plates, protein solutions and cocktail solutions).  Experiments are prepared using a liquid-handling system equipped with a 96 channel syringe head.  A 96-well source plate is loaded with 25 mL aliquots of as many as 96 different proteins; a second plate is prepared with aliquots of crystallization cocktails arrayed to match the proteins.  The 96 cocktail solutions are simultaneously aspirated from the source plate and dispensed into 4 replicate oil-filled 1536 well experiment plates. Each cocktail is delivered to the experiment plate16 times at volumes ranging from 0.15 to 0.60 mL.  The protein solutions are delivered in a similar manner into the experiment plates containing the cocktails as shown in Figure 1.  These deliveries are completed in less than one hour.  The four plates are separately incubated at temperatures of 4, 14, 23, and 37^oC.  These experiments are imaged weekly using the same plate imaging system that monitors the screening experiments.

Even in cases where DVR/T fails to identify chemical and environmental conditions that produce crystals suitable for X-ray diffraction, the method provides valuable solubility information that can be employed in second tier optimization protocols.  The DVR/T method landscapes the protein’s solubility as a function of the cocktail concentration, temperature and pH (Figure 1).  This multi-parametric sampling of solubility can be used to identify regions of supersaturation near the ideal (metastable) zone for seeding.  Seed crystals or microcrystalline precipitate can be recovered from supersaturated drops  and used for microseeding or macroseeding.  Undersaturated zones (clear drops) that are nearly supersaturated, based on the phase information provided by the DVR/T experiment, can be selected for seeding.
 
Temperature is a variable that is generally applicable to control the solubility of proteins.   Temperature can be used as a means to control the level of supersaturation in a  crystallization experiment and to produce diffraction-quality crystals.  DVR/T presents the sample’s solubility in such a way that it clearly identifies the range of temperatures to exploit for crystallization.  Data from DVR/T experiments highlight chemically identical experiments that appear clear, or show signs of phase separation by adjusting the temperature.  This information is clearly displayed in the outcomes of the DVR/T experiments (Figure 2).  We will use these data to design second tier optimization experiments.  Slowly adjusting the temperature of the experiment provides kinetic control of the approach to supersaturation.  The kinetic approach to supersaturation can be a critical variable to control during crystallization experiments.

Reference

Luft, J.R., Wolfley, J.R., Said, M.I., Nagel, R.M., Lauricella, A.M., Smith, J.L., Thayer, M.H., Veatch, C.K., Snell, E.H., Malkowski, M.G., DeTitta, G.T. (2007). Efficient Optimization of Crystallization Conditions by Manipulation of Drop Volume Ratio and Temperature. Protein Science 16, 1-8.  [PubMed]