Tables for
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 14.1, p. 293   | 1 | 2 |

Section 14.1.1. Introduction

B. W. Matthewsa*

aInstitute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon, Eugene, OR 97403, USA
Correspondence e-mail:

14.1.1. Introduction

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As is well known, the successful introduction of the method of isomorphous replacement by Green et al. (1954[link]) was the turning point in the subsequent development of protein crystallography as we now know it.

The idea that the phases of X-ray reflections from a protein crystal could be obtained by the introduction of heavy atoms into the crystal was not new, having been suggested by J. D. Bernal in 1939 (Bernal, 1939[link]). The isomorphous-replacement method was used as early as 1927 by Cork (1927[link]) in studying the alums. Bokhoven et al. (1951[link]) subsequently extended the method to the study of a noncentrosymmetric projection of strychnine sulfate, using what would now be termed the method of single isomorphous replacement. They also suggested that by using a double isomorphous replacement, a unique phase determination could be obtained, even for noncentrosymmetric reflections. The details of the double (or multiple) isomorphous-replacement method were worked out by Harker (1956[link]), who introduced the very useful concept of phase circles. Another contribution which was of great practical value, and which will provide the basis for much of the subsequent discussion, is the method introduced by Blow & Crick (1959[link]) for the treatment of errors in the isomorphous-replacement method. In addition to the determination of protein phases by the method of substitution with heavy atoms, it is now routine to supplement this information by utilizing the anomalous scattering of the substituted atoms. The underlying principles trace back to articles by Bijvoet (1954[link]), Ramachandran & Raman (1956[link]), and Okaya & Pepinsky (1960[link]). The first application of the anomalous-scattering method to protein crystallography was by Blow (1958[link]), who used the anomalous scattering of the iron atoms to determine phase information for a noncentrosymmetric projection of horse oxyhaemoglobin.

In the following discussion, we first review the classical method of phase determination by isomorphous replacement, then discuss the inclusion of single-wavelength anomalous-scattering data, and conclude by discussing the use of such data for heavy-atom location. Part of the review is based on Matthews (1970[link]).


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Bijvoet, J. M. (1954). Structure of optically active compounds in the solid state. Nature (London), 173, 888–891.Google Scholar
Blow, D. M. (1958). The structure of haemoglobin. VII. Determination of phase angles in the non-centrosymmetric [100] zone. Proc. R. Soc. London Ser. A, 247, 302–336.Google Scholar
Blow, D. M. & Crick, F. H. C. (1959). The treatment of errors in the isomorphous replacement method. Acta Cryst. 12, 794–802.Google Scholar
Bokhoven, C., Schoone, J. C. & Bijvoet, J. M. (1951). The Fourier synthesis of the crystal structure of strychnine sulphate pentahydrate. Acta Cryst. 4, 275–280.Google Scholar
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Harker, D. (1956). The determination of the phases of the structure factors of non-centrosymmetric crystals by the method of double isomorphous replacement. Acta Cryst. 9, 1–9.Google Scholar
Matthews, B. W. (1970). Determination and refinement of phases for proteins. In Crystallographic computing, edited by F. R. Ahmed, S. R. Hall & C. P. Huber, pp. 146–159. Copenhagen: Munksgaard.Google Scholar
Okaya, Y. & Pepinsky, R. (1960). New developments in the anomalous dispersion method for structure analysis. In Computing methods and the phase problem in X-ray crystal analysis, pp. 273–299. London: Pergamon Press.Google Scholar
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