User:WillowW/Crystallization

Methods
Crystallization of small molecules has traditionally followed three methods
 * Diffusion gradient- solubility or temperature
 * Concentration through evaporation
 * Sublimation (not recommended due to low-quality crystals).

Even though small molecules are easier to crystallize than macromolecules, there are many compounds reported that have failed to give diffraction quality crystals.

Crystallization of macromolecules is more difficult. Proteins must remain folded to allow crystallization, and folding is favored only within a relatively narrow range of solution conditions such as pH, temperature and organic solvent concentrations. Hence, the requirement for folding makes the range of crystallization solution conditions more limited than for small-molecule crystallization. Many methods exist to crystallize proteins, such as batch methods (mix the two solutions directly), simple dialysis (proteins can be un-"salted in"), concentration dialysis (proteins crystallize as their concentration is increased gradually), liquid diffusion, and the classic method of evaporation. The two most successful methods are the microbatch and vapor diffusion techniques. Concentrated solutions of the protein are mixed with various solutions, which typically consist of:
 * a buffer to control the pH of the experiment
 * a precipitating agent, to induce supersaturation (typically polyethylene glycols, salts such as ammonium sulphate, or organic alcohols).
 * other salts or additives, such as detergents or molecule co-factors

Vapor diffusion is exploited in most modern methods, such as the hanging drop and sitting drop methods (add Figure). In the hanging drop method, a small droplet of concentrated protein- and precipitant-containing solution is applied to a glass coverslip. The droplet is inverted and suspended above a larger reservoir of a similar solution lacking protein but containing a higher concentration of precipitant. In some cases, a thin coating of oil is added to the droplet to slow the vapor diffusion. A closed environment is formed containing the suspended droplet and reservoir. Over time, the droplet containing protein equilibrates with the larger reservoir as water is exchanged between the droplet and the reservoir, which gradually shrinks the droplet and concentrates the precipitant and protein in the droplet. In solutions of a favorable composition, the protein becomes supersaturated and crystal nuclei form, leading to crystal growth. This is the optimal outcome. However, it is more likely that the protein drop remains clear (the precipitating forces were too weak) or the protein forms a useless, amorphous dust as it precipitates ("crashes") out of solution (the precipitating forces were too strong). Protein crystallographers generally screen hundreds, even thousands of conditions before a suitable condition is found that leads to a crystal of suitable quality. As a rule of thumb, some useful structural detail can be gained from a crystal that diffracts with a resolution of better than 4 Ångströms (400 pm).

Many biomolecules of interest still have not been successfully crystallized. Imperfections in the crystal structure, caused by impurities, sample contamination, or multiple stable conformations of the subject protein can prevent the acquisition of atomic resolution images. Convection caused by temperature variations within the forming crystal can also cause imperfections, and one of the proposed scientific applications of the International Space Station is the growth of crystals, because convection is reduced in the free fall environment of an orbiting spacecraft.

Often one observes a crystal growing that is unfortunately not composed of the molecule of interest. For example, the salts of the crystallization solution used to crystallize a protein may themselves form a crystal. Such crystals can be discerned with dyes or checking the stiffness of the crystal. Protein crystals are pliant and absorb small dyes, being typically 50% water in their makeup. By contrast, salt crystals do not absorb dyes and are much more rigid.