Interactive Crystallomic - Molecules: Nucleation, Aggregation and Crystallization - page 226

arranged in a periodic and repeating fashion that extends in three (or two)
dimensions. Crystals may comprise 10 19 molecules. How many reliable
three-dimensional structures are available? The current holding of accurate
structures in the Protein Data Bank can be found online at www.rcsb.
org/pdb. There is indeed a steady increase in solved protein structures: on
February 15, 2005, 25 260 structures had been determined by X-ray diffrac-
tion (4376 determined by NMR); on August 1, 2006, there were 37 981
(4751 determined by NMR); on May 5, 2008, there were in total 42 342
structures determined by X-ray and 7150 by NMR. They are mainly water-
soluble proteins, peptides, viruses, protein/nucleic acid complexes, nucleic
acids or carbohydrates. In contrast, the output for crystallizing membrane
proteins or viruses has been regrettably low (36 proteins, Aoyama et al .,
2004; 50 viruses, Sedzik et al ., 2001). The limiting factor, referred to by
some researchers as “a bottleneck,” is simply to find the right physico-
chemical conditions in which dispersed, monomeric molecules can begin to
nucleate (aggregate) forming nuclei. Following this, spontaneous and steady
growth leads to a “large” crystal of size at least 0.1 mm in all dimensions.

Some proteins crystallize very easily and rapidly, for example lysozyme
(McPherson, 1990), while for others successful crystallization was only
achieved after 20 years (cytochrome c oxidase; Yoshikawa et al ., 1996).
In this chapter a new and rational approach to the process of crystal-
lizing biological molecules, which we have called interactive crystal-
lomic , is described. This strategy is affordable, efficient and economical,
and can easily be adopted by any laboratory struggling to overcome the
acute bottleneck of crystallization.

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Experimental Procedure — An Example

Pre-crystallization assumptions

Before carrying out these crystallization trials of virus-like particles, two
assumptions were made:

(i) use the same buffer for crystallization, in which virus-like particles

are purified (50 mM Tris, pH 7.4), and