Crystallography Tips

So you want to crystallize your protein? Here are some key factors:

Quantity
Purity
Homogeneity
Solubility
Activity

In general, a minimum of 10 mg of 95-99% pure protein is necessary to begin crystallization trials. Availability of a bacterial system for over-expression is quite valuable, but not required if you have other ways to produce large amounts of protein.

In addition to being pure, your protein must also be soluble at fairly high concentrations in aqueous buffers. For crystallization trials, an average size protein should be at approximately 10 mg/ml. Wide variations on necessary concentration have been reported, but 10 mg/ml is a good place to start.

For crystallization, your sample should be stable in minimal salt and buffer. Water is ideal, but 10-50 mM biological buffers are usually fine. If your sample needs salt to remain active or soluble, leave it in! Activity and structure in various solutions should be confirmed by whatever assays possible. Indirect structural information like that available from circular dichroism or light scattering can be quite helpful. If you work on small peptides (less than 30 residues) or integral membrane proteins, your needs can be quite different, and are not addressed in this general discussion.

Once you have pure, stable, and soluble samples, crystallization trials are quite easy. At this point, we suggest you contact the SBC for advice on how to proceed. The necessary supplies are generally inexpensive, except for a stereo microscope for examining crystallization drops. Hampton Research sells several crystallization kits and many helpful supplies.

Although time-consuming and sometimes tedious, screening your samples for crystals is not difficult. However, it may not be successful, no matter how many conditions are tried. The suggestions above regarding purity, concentration, and stability are your best leads to growing crystals. Anything that introduces heterogeneity into your sample should be avoided (for example, post-translational modifications like glycosylation).