Page 56 - Macromolecular Crystallography

CHAPTER 3

Automation of non-conventional

crystallization techniques for

screening and optimization

Naomi E. Chayen

3.1 Introduction 3.2 The basis of crystallization

The availability of high-quality crystals is cru- Crystallization is a phase transition phenomenon.
cial to the structure determination of proteins by Crystals grow from an aqueous protein solution
X-ray diffraction. It is still not understood why when the solution is brought into supersatura-
some proteins crystallize with ease while oth- tion (Ataka, 1993). Supersaturation is achieved
ers stubbornly refuse to produce suitable crystals. by varying the concentrations of precipitant, pro-
Producing high-quality crystals has always been tein and additives, pH, temperature, and other
the bottleneck to structure determination and with parameters (McPherson, 1999; Ducruix and Giegé,
the advent of structural genomics this problem is 1992; Ducruix and Giegé, 1999).
becoming increasingly acute. In spite of impres-
sive advances in throughput, the crystallization
problem has not been solved and better crystal- 3.2.1 The crystallization phase diagram
lization techniques need to be designed in order
to overcome this hurdle (Service, 2002; Chayen, The crystallization process can be illustrated by a
2004). Finding favourable conditions for crystal- phase diagram that shows which state (liquid, crys-
lization is usually achieved by screening of the talline, or amorphous solid [precipitate]) is stable
protein solution with numerous crystallizing agents under a variety of crystallization parameters. It pro-
in order to find ‘hits’ that indicate which condi- vides a means of quantifying the influence of the
tions may be suitable for crystal growth. Opti- parameters such as the concentrations of protein,
mization of the crystallization conditions is done precipitant(s), additive(s), pH, and temperature on
either by fine tuning of the parameters (precipi- the production of crystals. Hence phase diagrams
tant, pH, temperature, additives, etc.) involved, or form the basis for the design of crystal growth con-
by manipulation of the crystallization phase dia- ditions (McPherson, 1999; Ducruix and Giegé, 1992;
gram with the aim of guiding the experiment in Ducruix and Giegé, 1999; Chayen et al., 1996; and
the direction that will produce the desired results. references therein).
This chapter highlights a variety of non-standard Crystallization proceeds in two phases, nucle-
experimental methods of screening and optimiza- ation and growth. Nucleation, which is a prerequi-
tion techniques with a focus on those that have been site for crystallization, requires different conditions
automated and can be adapted to high-throughput from those of growth. Once the nucleus has
trials. formed, growthfollowsspontaneously(Ataka, 1993;

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