25] Overview of isomorphous replacement phasing

Abstract

This chapter discusses isomorphous replacement phasing. Multiple isomorphous replacement (MIR), multiwavelength anomalous diffraction (MAD), and molecular replacement (MR) are three popular methods for the phasing of macromolecular structures. With MR, an unknown structure can be solved by rotating and translating a known model into a new crystallographic system; therefore, it is not an ab initio phasing method. In the isomorphous replacement method, heavy-atom derivatives are prepared by soaking the native crystals in a buffer containing a heavy-metal compound or by cocrystallizing a protein with a heavy-metal compound. The use of a well-phased derivative in the initial refinement and then extension to other derivatives is always a preferred strategy. This procedure gives a better initial estimation of phases for the refinement and can result in better convergence. The use of phases improved by other methods for heavy-atom refinement gives better convergence of the refinement. Numerous computer programs are available for isomorphous replacement phasing. The combination of isomorphous replacement or anomalous scattering with phase-improvement methods helps in the automatic determination of macromolecular structures.