Absolute homogeneity is essential for optimal crystallization as well as crystallographic analysis. An awareness of possible heterogeneity combined with methods and efforts to avoid and remove heterogeneity in the sample preparation should be a priority. Possible sources of sample heterogeneity include the following.
• Presence, absence, or variation in a bound prosthetic group, ligand, cofactor, or metal ion
• Variation in composition of carbohydrate on a glycoprotein
• Unintentional proteolytic modification
• Oxidation of sulfhydryl groups
• Reaction with heavy metals
• Presence, absence, or variation in post-translational side chain modification (methylation, amidation, phosphorylation, glycosylation, or lipidation)
• Variation in amino of carboxy terminus, or modification of the terminus
• Variation in aggregation or oligomer state
• Conformational flexibility or instability due to the dynamic nature of the sample
• Incomplete or incorrect refolding or partial denaturation
• Combining different preps or purifications
Preparation and analysis of protein crystals. Alexander McPherson. 1982. 75-81. John Wiley & Sons.