Isomorphous replacement techniques

The isomorphous replacement method is a very old technique, used incidentally by Bragg to solve NaCl and KCl structures: it was later formulated in a more general way by Robertson (1935, 1936) and by Robertson and Woodward (1937). Its modern formulation is essentially due to Green et al. (1954) and to Bragg and Perutz (1954), who applied the method to haemoglobin. The technique has made possible the determination of the first three macromolecular structures, myoglobin, haemoglobin, and lysozyme. The approach may be summarized as follows. Suppose that the target structure is difficult to solve (e.g. it is a medium-sized structure, resistant to any phasing attempt, or it is a protein with bad data resolution) and we want to adopt isomorphous replacement techniques. Then we should perform the following steps: (a) Collect the diffraction data of the target structure; in the following we will suppose that it is the native protein. (b) Crystallize a new compound in which one or more heavy atoms are incorporated into the target structure. This new compound is called derivative. (c) Check if the operations in (b) heavily disturb the target structure. If not, the derivative is called isomorphous; then, only local (in the near vicinity of the binding site) structural modifications are induced by the heavy atom addition. Non-isomorphous derivative data are useless. (d) Use the two sets of diffraction data, say set {|FP|} of the target structure and set {|Fd|} of the isomorphous derivative, to solve the target structure. The above case is referred to as SIR (single isomorphous replacement). The reader should notice that redundant experimental information is available; indeed, two experimental sets of diffraction data relative to two isomorphous structures may be simultaneously used for solving the native protein. The redundancy of the experimental information allows crystal structure solution even if data resolution is far from being atomic (e.g. also when RES is about 3 or 4 Å, and even more in lucky cases). Imperfect isomorphism may hinder crystal structure solution. Then, more derivatives may be prepared; their diffraction data may be used in a combined way and may more easily lead the phasing process to success.