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

International Tables for Crystallography (2006). Vol. F, ch. 12.1, p. 247   | 1 | 2 |

Section 12.1.1. Introduction

D. Carvin,a S. A. Islam,b M. J. E. Sternbergb and T. L. Blundellc*

aBiomolecular Modelling Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, England,bInstitute of Cancer Research, 44 Lincoln's Inn Fields, London WC2A 3PX, England, and cDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, England
Correspondence e-mail:  tom@cryst.bioc.cam.ac.uk

12.1.1. Introduction

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The traditional method of multiple isomorphous replacement (MIR) was introduced by Perutz and co-workers in 1954 (Green et al., 1954[link]) and is often enhanced by anomalous scattering (MIRAS) [see Blundell & Johnson (1976[link]) for a review]. The method remains popular for solution of the phase problem in the absence of the structure of a close homologue, although the use of multiple anomalous dispersion is likely to increase in the coming years (Hendrickson, 1985).

Protein crystals comprise an open lattice of protein molecules with solvent occupying the channels and spaces which normally comprise between 30 and 80% of the crystal volume. The preparation of a useful derivative requires the binding of a heavy atom to a specific position, usually on the protein surface, for example by the displacement of a lighter solvent molecule or an ion, without distorting the protein or crystal lattice.

Ideally, rational selection of suitable heavy-atom reagents requires a comprehensive knowledge and understanding of the crystalline structure of the protein. Normally, this information is unavailable since it is the objective of the crystal structure analysis! Nevertheless, the sequence and mechanism of action may suggest which heavy-atom reagents might be employed. There are reports in the literature of many attempts to make synthetic analogues of specific amino acids, by substituting selenium for sulfur residues in a chemically synthesized polypeptide or by removing an amino-terminal residue by the Edman technique and replacing it with an amino acid modified by a heavy atom [see Blundell & Johnson (1976[link]) for a review]. Alternatively, analogues of the substrate of an enzyme or carrier protein can sometimes be modified with a heavy atom; however, this will disturb the active site, which is usually the region of greatest interest to the structural biologist. Such methods have not proved very useful and will not be described further here. Most proteins studied now are recombinant; site-directed mutagenesis can replace methionines in the sequence, which occur on average once every fifty residues, by selenomethionines (Hendrickson et al., 1990[link]) or more recently by telluromethionines (Budisa et al., 1997[link]). Such approaches have revolutionized macromolecular crystallography through the use of anomalous-dispersion techniques, but have yet to provide a very efficient method of introducing atoms heavier than selenium into proteins.

Thus, the vast majority of successful heavy-atom derivatives employed in crystallographic analyses are obtained on a trial-and-error basis. In earlier studies, the protein was often covalently modified, purified and characterized before crystallization. There are some useful covalent modifications, for example, the reaction of mercury with the sulfhydryl groups of cysteinyl side chains and the iodination of tyrosyl side chains. The replacement of a metal-ion cofactor, such as calcium or zinc, can also give a useful derivative. However, pre-reaction of the protein often gives rise to conformational changes in the protein, and crystallization frequently occurs in a different or non-isomorphous form.

Most heavy-atom derivatives are produced by direct soaking of the crystals in a solution of the heavy-atom compound. With this approach, heavy-atom substitution patterns tend to be complex, with sites frequently only partially occupied. The specificity is often determined by entropic factors. Thus, sites between molecules in the crystal lattice, or between several different side chains brought together by the tertiary structure, may bind the metal ion, even if the side chains individually do not have strong affinity for the metal. Chelation is entropically driven, and bonds may form with unusual protein ligands, a major factor causing lack of specificity.

Blake (1968[link]) reviewed the data available for heavy-atom binding to proteins and suggested some generalizations. These were extended in a comprehensive review of protein heavy-atom derivatives (Blundell & Johnson, 1976[link]; Blundell & Jenkins, 1977[link]) which analysed the dependence of reactivity on protein side chain identity, nature of the reagent, pH, concentration, buffer etc. Over the past two decades, there have been discussions of the binding of some particular metal ions, but there have been no comprehensive analyses. Furthermore, protein–heavy-atom interactions have not often been fully described in publications of protein crystallographic analyses, and, in any case, the information has not been available in a format that could be used for systematic computer-based analysis.

We have now collected, either from the literature or directly from protein crystallographers, information on the preparation and characterization of heavy-atom derivatives of protein crystals. We have defined heavy atoms as those with an atomic weight greater than that of rubidium. We have assembled the information in the form of a data bank (Carvin et al., 1991[link]; Islam et al., 1998[link]) in which the coordinate data for the heavy-atom positions are compatible with the crystallographic data in the Protein Data Bank (Bernstein et al., 1977[link]). The data bank contains a wealth of information and provides the basis for further, more detailed analyses of heavy-atom binding to proteins. The information can be directly accessed and should be useful to protein crystallographers seeking to improve their success in preparing heavy-atom derivatives for isomorphous replacement and anomalous dispersion.

In this chapter we provide an introduction to the data bank and we review strategies that can be adopted in the preparation of heavy-atom derivatives of protein crystals for use in MIRAS.

References

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Blake, C. C. F. (1968). The preparation of isomorphous derivatives. In Advances in protein chemistry, Vol. 23, pp. 59–120. New York and London: Academic Press.Google Scholar
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