Structure Determination Using Isomorphous Replacement

The determination of the three-dimensional structure of molecules using single crystal X-ray diffraction techniques requires the measurement of amplitudes and the calculation of phases for each diffraction point (maximum). Although amplitudes can be directly measured from diffracting crystals, phases are indirectly determined, because there are no lenses that can bend and focus X-rays. Thus, methods were developed to calculate phases from the intensities of the diffracted waves. Isomorphous replacement is the most widely used method for ab initio phase determination of macromolecules. Its first successful application to large biological molecules was undertaken in 1954 by Perutz and coworkers (1 ) while studying hemoglobin. Since then, this method has played a central role in the determination of almost all unique protein and nucleic acid structures, and it is likely to retain such a role in the foreseeable future.