84 INSTRUMENTAL METHODS
3.3.2 Crystallization and Crystal Habits,
Protein structure determination using X - ray crystallography fi rst requires the
production of suitable crystals. Protein crystallization remains mainly a trial -
and - error procedure complicated by impurities contaminating the selected
protein, by the poorly understood process of crystal nucleation, and by other
unknown factors. Protein purity requires not only that other compounds be
absent but also that all molecules of the protein have the same surface proper-
ties (charge distribution) as the latter affects crystal packing of the molecules.
After multistep purifi cation procedures, the protein is dissolved in a suitable
solvent (usually water – buffer mixtures for pH control) and the solution is
brought to supersaturation so that nuclei for crystal growth appear. To avoid
continued nucleation that will result in crystals too small for diffraction and
to foster crystal growth, supersaturation must be decreased at this point. This
is usually achieved by raising the temperature. The next step involves addition
of a precipitant such as polyethyleneglycol (PEG), salt, or an organic solvent
and perhaps adjustment of pH.
Crystallization techniques include liquid – liquid diffusion, several types of
vapor diffusion, and dialysis through a membrane. Liquid – liquid diffusion of
protein and precipitant solutions may conveniently take place in a melting
point capillary tube using approximately 5 μ l of each solution. The solution of
higher density, which may be either the protein solution or the precipitant
solution, is added to the capillary tube fi rst using a syringe. The other solution
is layered onto the top of the fi rst forming a sharp boundary. Slow diffusion
of the layers may produce suitable crystals, although it may be necessary to
test many variations of solvent, solute concentration, and diffusing solvent
before the desired crystal size and purity is attained. The most common vapor
diffusion method is called the hanging drop method. Drops containing 3 – 10 μ l
of protein and precipitant solutions are placed on a siliconized (to prevent
spreading of the drop) microscope glass cover slip. The slip is placed upside
down over a depression in a tray. The depression is partially fi lled with pre-
cipitant solution. Diffusion of vapors from or to the precipitant solution may
result in crystal formation given the same caveats as mentioned above for
solvent diffusion. Many different membrane dialysis techniques are available.
The advantage of this technique is that the protein solution is confi ned within
a membrane allowing different precipitant solutions outside the membrane to
be tried sequentially. Gilliland and Ladner 12a have published a review of crys-
tallization techniques, and Gilliland 12b has assembled a database of macromol-
ecule crystal and crystallization data. To be suitable for X - ray crystallography,
a crystal is usually required to be≥ 0.1 mm in its longest dimension.
The fl at faces of crystals refl ect the regular packing of atoms, molecules,
or ions within the crystalline structure, resulting in anisotropic (directional)
behavior. Because of the regular arrangement of the crystal ’ s atoms, molecules,
or ions in planes with fi xed angles and distances between the planes, single
crystals will diffract X - rays. As an example, placing a crystal having a fourfold