286 7.5  Making Crystals

Many functional biomolecular complexes may be formed from multiple separate

components. Obtaining crystals from these is harder since it requires not only a mixture

of highly pure separate components but also one in which the relative stoichiometry of the

components to each other is tightly constrained. Finding optimum temperature and pH

conditions that avoid premature precipitation in the separate components is a key challenge

often requiring significant experimental optimization. The use of microorganisms such as

bacteria and unicellular eukaryotes to grow such crystals has shown recent promise, since

the small volume of these cells can result in very concentrated intracellular protein concen­

tration. Crystals for viral capsids (see Chapter 2) have been generated in this way, with the

caveat that the crystal size will be limited to just a few microns length due to the small size

of the cells used.

7.5.3  TREATMENT AFTER CRYSTALLIZATION

The precision of an x-​ray crystal diffraction pattern is affected significantly by the homo­

geneity of the crystal, and its size. Controlled, gradual dehydration of crystals can result in

an ultimate increase in crystal size, for example, using elevated concentration levels of PEG

to draw out the water content, which in some cases can alter the shape of the crystal unit

cell, resulting in more efficient packing in a larger crystal structure. Also, small seed crystals

placed in the undersaturated solution can be efficient sites for nucleation of larger growing

larger crystals.

The use of crystallization robots has significantly improved the high-​throughput nature

of crystallization. These devices utilize vapor diffusion methods to automate the process of

generating multiple crystals. They include multiple arrays of microwell plates, resulting in

several tens of promising crystals grown in each batch under identical physical and chemical

conditions using microfluidics (see later in this chapter). These methods also utilize batch

screening methods to indicate the presence of promising small crystals that can be used as

seed crystals. The detection of such small crystals, which may have a length scale of less than

a micron, by light microscopy is hard but may be improved by UV excitation and detection

of fluorescence emission from the crystals or by using polarization microscopy. Second har­

monic imaging (see Chapter 4) can also be used in small crystal identification, for example, in

a technique called second-​order nonlinear optical imaging of chiral crystals. The use of such

high-​throughput technologies in crystallization with robotized screening has enabled the

selection of more homogeneous crystals from a population.

7.5.4  PHOTONIC CRYSTALS

A special type of crystal, which can occur naturally both in living and nonliving matter and

also which can be engineered synthetically for biophysical applications, is photonic crystals.

Photonic crystals are spatially periodic optical nanostructures that perturb the propagation

of transmitted photons. This is analogous to the perturbation of electrons in ionic crystal

structures and semiconductors, for example, there are certain energy levels that are forbidden

in terms of propagation of photons, in the same manner that there are forbidden energy levels

for electron propagations in certain spatially periodic solids.

Photonic crystals are spatially periodic in terms of dielectric constant, with the period­

icity being comparable to the wavelength of visible or near visible light. This results in dif­

fractive effects only for specific wavelengths of light. An allowed wavelength of propagation

is a mode, with the summation of several modes comprising a band. Disallowed energy bands

imply that photons of certain wavelengths will not propagate through the crystal, barring

small quantum tunneling effects, and are called “photonic bandgaps.”

Natural nonbiological photonic crystals include various gemstones, whereas biological

photonic crystals include butterfly wings. Butterfly wings are composed of periodic scales

made from fibrils of a protein called chitin combined with various sugar molecules in a matrix

of other proteins and lipids that, like all crystals, appear to have many self-​assembly steps