The toughest obstacle in determining the three-dimensional structures of macromolecules is obtaining crystals of the molecule in question — protein or nucleic acid. The process of crystallization remains largely an art form. In favorable cases, success usually requires very many experimental trials under different conditions. In unfavorable cases, crystals are never obtained. The reasons for failure are sometimes understandable — e.g. flexibility or heterogeneity — but are just as often unknown. Numerous strategies have been developed to improve the chances of success. Some of these are based on the principle that it is more fruitful to create variations on the macromolecule of interest, and to attempt crystallizing these multiple variants, rather than performing an infinitude of different trials on a single molecular construct.

One family of approaches, which we refer to as ‘synthetic symmetrization’ (Banatao, et al., 2006), seeks to make a series of distinct symmetric constructs from a starting macromolecule that is otherwise asymmetric (i.e. a monomer) (see Figure at right). If the starting molecule can be made symmetric in a variety of distinct ways (e.g. by dimerization at different contact points), then each of the resulting constructs will enjoy distinct opportunities for packing in crystalline arrangements. In addition, the presence of symmetry, by itself, confers some advantage in crystallization.

The first approach demonstrated for synthetic symmetrization was disulfide bonding following the introduction of single cysteine residues at surface positions in a protein molecule. Applying the method to lysozyme led to six new crystal forms of that protein (Banatao, et al., 2006). The cysteine cross-linking approach was subsequently used to determine the structure of a protein of previously unknown structure, endoglucanase A (Forse, et al., 2011).

A potentially more versatile strategy based on metal binding was developed by three graduate students (Laganowsky, Zhao, Soriaga, et al., 2011), adapting ideas introduced by Akif Tezcan’s laboratory. Two histidine residues are introduced at positions i and i+4 in a region of the sequence predicted to constitute a surface-exposed helix. During crystallization, addition of metal (i.e. nickel, zinc, or copper) leads to formation of dimers (or sometimes other oligomeric species). Distinct constructs, with histidines introduced at different positions, have distinct crystal packing opportunities and give rise to new crystal forms. The metal-based symmetrization approach has so far been demonstrated with lysozyme and maltose binding protein as model systems.