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.
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|
|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|
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.