282 7.5  Making Crystals

resolution. There are therefore important aspects to the practical methods for generating

biomolecular crystals, which we discuss here.

7.5.1  BIOMOLECULE PURIFICATION

The first step in making biomolecule crystals is to purify the biomolecule in question. Crystal

manufacture ultimately requires a supersaturated solution of the biomolecule (meaning a

solution whose effective concentration is above the saturation concentration level equivalent

to the concentration in which any further increase in concentration results in biomolecules

precipitating out of solution). This implies generating high concentrations equivalent in prac­

tice to several mg mL⁻¹.

Although crystals can be formed from a range of biomolecule types, including sugars and

nucleic acids, the majority of biomolecule crystal structures that have been determined relate

to proteins, or proteins interacting with another biomolecule type. The high purity and con­

centration required ideally utilizes molecular cloning of the gene coding for the protein in a

plasmid to overexpress the protein. However, often a suitable recombinant DNA expression

system is technically too difficult to achieve, requiring less ideal purification of the protein

from its native cell/​tissue source. This requires a careful selection of the best model organism

system to use to maximize the yield of protein purified. Often bacterial or yeast systems are

used since they are easy to grow in liquid cultures; however, the quantities of protein required

often necessitate the growth of several hundred liters of cells in culture.

The methods used for the extraction of biomolecules from the native source are classical

biochemical purification techniques, for example, tissue homogenization, followed by a series

of fractionation precipitation stages. Fractionation precipitation involves altering the solu­

bility of the biomolecule, most usually a protein, by changing the pH and ionic strength of

the buffer solution. The ionic strength is often adjusted by addition of ammonium sulfate at

high concentrations of ~2.0 M, such that above certain threshold levels of ammonium sulfate,

a given protein at a certain pH will precipitate out of a solution, and so this procedure is also

referred to as ammonium sulfate precipitation.

At low concentrations of ammonium sulfate, the solubility of a protein actually increases

with increasing ammonium sulfate, a process called “salting in” involving an increase in the

number of electrostatic bonds formed between surface electrostatic amino acid groups and

water molecules mediated through ionic salt bridges. At high levels of ammonium sulfate,

the electrostatic amino acid surface residues will all eventually be fully occupied with salt

bridges and any extra added ammonium sulfate results in the attraction of water molecules

away from the protein, thus reducing its solubility, known as salting out. Different proteins

salt in and out at different threshold concentrations of ammonium sulfate; thus, a mixture

of different proteins can be separated by centrifuging the sample to generate a pellet of the

precipitated protein(s) and then subject either the pellet or the suspension to further bio­

chemical processing—​for example, to use gel filtration chromatography to further separate

any remaining mixtures of biomolecules on the basis of size, shape, and charge, etc., in add­

ition to methods of dialysis (see Chapter 6). Ammonium sulfate can also be used in the final

stage of this procedure to generate a high concentration of the purified protein, for example,

to salt out, then resuspend the protein, and dissolve fully in the final desired pH buffer for

the purified protein to be crystallized. Other precipitants aside from ammonium sulfate can

be used depending on the pH buffer and protein, including formate, ammonium phosphate,

the alcohol 2-​propanol, and the polymer polyethylene glycol (PEG) in a range of molecular

weight value from 400 to 8000 Da.

7.5.2  CRYSTALLIZATION

Biomolecule crystallization, most typically involving proteins, is a special case of a thermo­

dynamic phase separation in a nonideal mixture. Protein molecules separate from water in

solution to form a distinct, ordered crystalline phase. The nonideal properties can be modeled