Section 4.1.4. How to crystallize a new macromolecule

The first concern is to obtain a macromolecular sample of highest quality; second, to collate all biochemical and biophysical features characterizing the macromolecule in order to design the best crystallization strategy; and finally, to establish precise protocols that ensure the reproducibility of experiments. It is also important to clean and sterilize by filtration (over 0.22 µm porosity membranes) all solutions in contact with pure macromolecules to remove dust and other solid particles, and to avoid contamination by microbes. Inclusion of sodium azide in crystallizing solutions may discourage invasive bacteria and fungi. In vapour-diffusion assays, such contamination can be prevented by simply placing a small grain of thymol in the reservoir. Thymol, however, can occasionally have specific effects on crystal growth (Chayen et al., 1989) and thus may serve as an additive in screenings as well.

Crystallization requires bringing the macromolecule to a supersaturated state that favours nucleation. Use of phase diagrams may be important for this purpose (Haas & Drenth, 1998; Sauter, Lorber et al., 1999). If solubilities or phase diagrams are unavailable, it is nevertheless important to understand the correlation between solubility and the way supersaturation is reached in the different crystallization methods (see Fig. 4.1.2.1). In dialysis, the macromolecule concentration remains constant during equilibration. The initial concentration of the crystallizing agent in the exterior solution leaves the macromolecule in an undersaturated state. With increasing concentration of the agent in the exterior solution, a state of supersaturation can be attained, leading to crystallization or precipitation. In a vapour-diffusion experiment, where the concentration of crystallizing agent in the reservoir exceeds that in the drop, the macromolecule will begin to concentrate from an undersaturated to a supersaturated state, with both macromolecule and crystallizing-agent concentrations increasing. Crystals appear in the metastable region. For crystals that appear first, the trajectory of equilibration is complex and the remaining concentration of macromolecule in solution will converge towards a point located on the solubility curve. In batch crystallization using a closed vessel, three situations can occur: if the concentration of the macromolecule is undersaturated, crystallization never occurs (unless another parameter such as temperature is varied); if it belongs to the supersaturated region between solubility and precipitation curves, crystals can grow until the remaining concentration of the macromolecule in solution equals its solubility; if supersaturation is too high, the macromolecule precipitates immediately, although in some cases, crystals can grow from precipitates by Ostwald ripening (Ng et al., 1996).

The concept of purity assumes a particular importance in crystallogenesis (Giegé et al., 1986; Rosenberger et al., 1996), even though some macromolecules may crystallize readily from impure solutions (Judge et al., 1998). In general, macromolecular samples should be cleared of undesired macromolecules and small molecules and, in addition, should be pure in terms of sequence integrity and conformation. Contaminants may compete for sites on growing crystals and generate growth disorders (Vekilov & Rosenberger, 1996), and it has been shown that only p.p.m. amounts of foreign molecules can induce formation of non-specific aggregates, alter macromolecular solubility, or interfere with nucleation and crystal growth (McPherson et al., 1996; Skouri et al., 1995). These effects are reported to be reduced in gel media (Hirschler et al., 1995; Provost & Robert, 1995).

Microheterogeneities in purified samples can be revealed by analytical methods, such as SDS–PAGE, isoelectric focusing, NMR and mass spectroscopy. Although their causes are multiple, the most common ones are uncontrolled fragmentation and post-synthetic modifications. Proteolysis represents a major difficulty that must be overcome during protein isolation. Likewise, nucleases are a common cause of heterogeneity in nucleic acids, especially in RNAs that are also sensitive to hydrolytic cleavage at alkaline pH and metal-induced fragmentation. Fragmentation can be inhibited by addition of protease or nuclease inhibitors during purification (Lorber & Giegé, 1999). Conformational heterogeneity may originate from ligand binding, intrinsic flexibility of the macromolecule backbones, oxidation of cysteine residues, or partial denaturation. Structural homogeneity may be improved by truncation of the flexible parts of the macromolecule under study (Price & Nagai, 1995; Berne et al., 1999).

Preparation of solutions for crystallization experiments should follow some common rules. Stocks should be prepared with chemicals of the purest grade dissolved in double-distilled water and filtered through 0.22 µm membranes. The chemical nature of the buffer is an important parameter, and the pH of buffers, which must be strictly controlled, is often temperature-dependent, especially that of Tris buffers. Commercial PEG contains contaminants, ionic (Jurnak, 1986) or derived from peroxidation, and thus repurification is recommended (Ray & Puvathingal, 1985).

Mother liquors are defined as the solutions that contain all compounds (buffer, crystallizing agent, etc.) at the final concentration for crystallization except the macromolecule. Samples of macromolecules often contain quantities of salt of unknown composition, and it is therefore wise to dialyse new batches against well characterized buffers. Whatever the crystallization method used, it almost always requires a high concentration of macromolecule. This may imply concentration steps using devices operating under nitrogen pressure, by centrifugation, or by lyophilization (notice that lyophylization may denature proteins and that non-volatile salts also lyophilize and will accumulate). Dialysis against high-molecular-weight PEG may also be used. During concentration, pH and ionic strength may vary and, if not kept at the appropriate values, denaturation of samples may occur.

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.