Section 4.1.4.4. Strategic concerns: a summary

Homogeneity : Perhaps the most important property of a system to be crystallized is its purity. Crystallization presupposes that identical units are available for incorporation into a periodic lattice. If crystallization fails, reconsidering purification protocols often helps achieve success.

Stability : No homogeneous molecular population can remain so if its members alter their form, folding, or association state. Hence, it is crucial that macromolecules in solution are not allowed to denature, aggregate, or undergo conformational changes.

Solubility : Before a molecule can be crystallized, it must be solubilized. This means creation of monodisperse solutions free from aggregates and molecular clusters. Solubility and crystallizability strongly depend on substances (organic solvents and PEGs) that reduce the ionic strength of the solution (Papanikolau & Kokkinidis, 1997).

Supersaturation : Crystals grow from systems displaced from equilibrium so that restoration requires formation of the solid state. Thus, the first task is to find ways to alter the properties of the crystallizing solutions, such as by pH or temperature change, to create supersaturated states.

Association : In forming crystals, molecules organize themselves through self-association to produce periodically repeating three-dimensional arrays. Thus, it is necessary to facilitate positive molecular interactions while avoiding the formation of precipitate or unspecific aggregates, or phase separation.

Nucleation : The number, size and quality of crystals depend on the mechanisms and rates of nuclei formation. In crystallization for diffraction work, one must seek to induce limited nucleation by adjustment of the physical and chemical properties of the system.

Variety : Macromolecules may crystallize under a wide spectrum of conditions and form many polymorphs. Thus, one should explore as many opportunities for crystallization as possible and explore the widest spectrum of biochemical, chemical and physical parameters.

Control : The ultimate value of any crystal is dependent on its perfection. Perturbations of the mother liquor are, in general, deleterious. Thus, crystallizing systems have to be maintained at an optimal state, without fluctuations or shock, until the crystals have matured.

Impurities : Impurities can contribute to a failure to nucleate or grow quality crystals. Thus, one must discourage their presence in the mother liquor and their incorporation into the lattice.

Perfection : Crystallization conditions should be such as to favour crystal perfection, to minimize defects and high mosaicity of the growing crystals, and to minimize internal stress and the incorporation of impurities. Predictions from crystal-growth theories may help to define such conditions (Chernov, 1997b, 1999).

Preservation : Macromolecular crystals may degrade and lose diffraction quality upon ageing. Thus, once grown, crystals may be stabilized by temperature change, addition of more crystallizing agent, or by some other suitable alteration in the mother liquor.