Growing crystals Basic procedure

Crystals of an inorganic substance can often be grown by making a hot, saturated solution of the substance and then slowly cooling it. Polar organic compounds can sometimes be crystallized by similar procedures or by slow precipitation from aqueous solutions by addition of organic solvents. If you work with proteins, just the mention of these conditions probably makes you 

One widely used technique is vapor diffusion, in which the proteinlprecipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir whose precipitant concentration is optimal for producing crystals. An example of this technique is the hanging-drop method (Fig. 3.2). 

Less than 25 pL of the solution of purified protein is mixed with an equal amount of the reservoir solution, giving precipitant concentration about 50% of that required for protein crystallization. This solution is suspended as a droplet underneath a cover glass, which is sealed onto the top of the reservoir grease. Because the precipitant is the major solute present, vapor diffusion in this closed system results in net transfer of water from the protein solution to the reservoir, until the precipitant concentration is the same in both solutions. Because the reservoir is much larger than the protein solution, the final concentration of the precipitant in the protein solution is nearly equal to that in the reservoir. When the system comes to equilibrium, net transfer of water ceases, and the protein solution is maintained at the optimal precipitant con- 

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For a number of years now we have been involved in crystallization of bacterial ribosomal particles. From the very beginning of our studies, the crucial need for a stable, very intense, and perfectly focussed synchrotron beam was evident, even for preliminary and basic information (e.g. whether crystals diffract at all). Thus our studies have always been dependent on the availability of synchrotron beam time and hampered by only partial (and very occasional) feedback to assess our experimental procedures for growing bacteria, preparing the ribosomes and obtaining crystals. 

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