Crystallization of biological macromolecules composed of many thousands of different atoms, bound together with many degrees of freedom, is a complex task. Confounding this many variables and factors influencing the crystallization experiment (Tables 1 & 2).1-3

This extensive number of variables confounded with typically limited sample material negates a precise and reasoned strategy typically applied to a scientific problem. Instead, crystallization is often a matter of searching, as systematically as possible, through crystallization experiments, to identify those variables key to success, as well as their ranges. Initially, one employs crystallization screening, typically to identify a hit, an association of variables that produces a crystal. In some instances this will produce crystals with the desired characteristics. More often than not, a series of successive experiments, termed optimization, will need to be carried out, in order to produce crystals with the desired properties, be it for structural biology, purification, formulation, or the delivery of a biological therapeutic.

Table 1.
Biochemical & Chemical variables that could or do affect protein crystal growth

Purity of the sample Genetic modification
Conformational flexibility of the sample Symmetry of the molecule
Homogeneity of the sample Stability and level of denaturation of the sample
pH and buffer Isoelectric point
Type and concentration of the precipitant (reagent) His tags and other purification tags - presence or absence
Concentration of the sample Thermal stability
Purity of the sample pH stability
Additives, co-factors, ligands, inhibitors, effectors, and excipients History of the sample
Chaotropes Proteolysis
Detergents Microbial contamination
Metals Storage of the sample
Ionic strength Handling of the sample and associate cleanliness
Reducing or oxidizing agents Anion and cation type and concentration
Source of the sample Degree of relative supersaturation
Presence of amorphous or particulate material Initial and final concentration of the reagent
Post-translational modifications Path and rate of equilibration
Chemical modifications  


Table 2.
Physical variables that could or do affect protein crystal growth

Temperature Electric and magnetic fields
Rate of equilibration Surface of the crystallizatioon device
Method of crystallization Viscosity of the reagent
Gravity, convection, and sedimentation Heterogeneous and epitaxial nucleants
Vibration and sound Geometry of crystallization device
Volume of the sample and reagent Time
Pressure Dielectric property of the reagent


References and Readings
1. Preparation and Analysis of Protein Crystals, McPherson, A. (1982). New York: John Wiley & Sons.
2. Current approaches to macromolecular crystallization. McPherson, A. (1990). Eur. J. Biochem. 189, 1-23.
3. Crystallization of Biological Macromolecules, McPherson, A. (1999). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.