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

International Tables for Crystallography (2006). Vol. F, ch. 4.3, p. 100   | 1 | 2 |

Section 4.3.1. Introduction

D. R. Daviesa* and A. Burgess Hickmana

aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0560, USA
Correspondence e-mail:

4.3.1. Introduction

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There is accelerating use of protein engineering by protein crystallographers. Site-directed mutations are being used for a variety of purposes, including solubilizing the protein, developing new crystal forms, providing sites for heavy-atom derivatives, constructing proteolysis-resistant mutants and enhancing the rate of crystallization. Traditionally, if the chosen protein failed to crystallize, a good strategy was to examine a homologous protein from a related species. Now, the crystallographer has a variety of tools for directly modifying the protein according to his or her choice. This is owing to the development of techniques that make it easy to produce a large number of mutant proteins in a timely manner (see Chapter 3.1[link] ).

The relevance to macromolecular crystallography of these mutational procedures rests on the assumption that the mutations do not produce conformation changes in the protein. It is often possible to measure the activity of the protein in vitro and, therefore, test directly whether mutation has affected the protein's properties. Several observations suggest that changes of a small number of surface residues can be tolerated without changing the three-dimensional structure of a protein. The work on haemoglobins demonstrated that mutant proteins generally have similar topologies to the wild type (Fermi & Perutz, 1981[link]). The systematic study of T4 phage lysozyme mutants by the Matthews group (Matthews, 1993[link]; Zhang et al., 1995[link]) has confirmed and significantly extended these studies and has provided a basis for mutant design. This work revealed that, for monomeric proteins, `Substitutions of solvent-exposed amino acids on the surfaces of proteins are seen to have little if any effect on protein stability or structure, leading to the view that it is the rigid parts of proteins that are critical for folding and stability' (Matthews, 1993[link]). It was also concluded that point mutants do not interfere with crystallization unless they affect crystal contacts. The corollary from this is that if the topology of the protein is known from sequence homology with a known structure, the residues that are likely to be located on the surface can be defined and will provide suitable targets for mutation. Fortunately, even in the absence of such information, it is usually possible to make an informed prediction of which residues (generally charged or polar) will, with reasonable probability, be found on the surface.

Here, we shall outline some of the procedures that have been used successfully in protein crystallography. We have tried to provide representative examples of the variety of techniques and creative approaches that have been used, rather than attempting to assemble a comprehensive review of the field. The identification of appropriate references is a somewhat unreliable process, because information regarding these attempts is usually buried in texts; we apologize in advance for any significant omissions.

There have been several reviews on the general topic of the application of protein engineering to crystallography. An overview of the subject is provided by D'Arcy (1994[link]), while Price & Nagai (1995[link]) `focus on strategies either to obtain crystals with good diffraction properties or to improve existing crystals through protein engineering'. In addition to attempts at a rational approach to protein engineering, it is worth emphasizing the role of serendipity in achieving the goal of diffraction-quality crystals. One example is given by the structure of GroEL (Braig et al., 1994[link]), where better crystals were obtained by the accidental introduction of a double mutation, which arose from a polymerase error during the cloning process. The second example is provided by the search for crystals of the complex between the U1A spliceosomal protein and its RNA hairpin substrate (Oubridge et al., 1995[link]). Initially, only poorly diffracting crystals (7–8 Å) could be obtained, which were similar in morphology to those of the protein alone. A series of mutations were made, designed to improve the crystal contacts, but the end result was a new crystal form that diffracted to 1.7 Å.

Dasgupta et al. (1997[link]), in an informative review, have compared the contacts formed between molecules in crystal lattices and in protein oligomerization. They found that there are more polar interactions in crystal contacts, while oligomer contacts favour aromatic residues and methionine. Arginine is the only residue prominent in both, and for a protein that is difficult to crystallize, they recommend replacing lysine with arginine or glutamine. Carugo & Argos (1997[link]) also examined crystal-packing contacts between protein molecules and compared these with contacts formed in oligomers. They observed that the area of the crystal contacts is generally smaller, but that the amino-acid composition of the contacts is indistinguishable from that of the solvent-accessible surface of the protein and is dramatically different from that observed in oligomer interfaces.


Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L. & Sigler, P. B. (1994). The crystal structure of the bacterial chaperonin GroEL at 2.8 Å. Nature (London), 371, 578–586.
Carugo, O. & Argos, P. (1997). Protein–protein crystal-packing contacts. Protein Sci. 6, 2261–2263.
D'Arcy, A. (1994). Crystallizing proteins – a rational approach? Acta Cryst. D50, 469–471.
Dasgupta, S., Iyer, G. H., Bryant, S. H., Lawrence, C. E. & Bell, J. A. (1997). Extent and nature of contacts between protein molecules in crystal lattices and between subunits of protein oligomers. Proteins, 28, 494–514.
Fermi, G. & Perutz, M. F. (1981). Atlas of molecular structures in biology, Vol. 2. Oxford: Clarendon Press.
Matthews, B. W. (1993). Structural and genetic analysis of protein stability. Annu. Rev. Biochem. 62, 139–160.
Oubridge, C., Ito, N., Teo, C.-H., Fearnley, I. & Nagai, K. (1995). Crystallisation of RNA-protein complexes II. The application of protein engineering for crystallisation of the U1A protein–RNA complex. J. Mol. Biol. 249, 409–423.
Price, S. R. & Nagai, K. (1995). Protein engineering as a tool for crystallography. Curr. Opin. Biotech. 6, 425–430.
Zhang, X., Wozniak, J. A. & Matthews, B. W. (1995). Protein flexibility and adaptability seen in 25 crystal forms of T4 lysozyme. J. Mol. Biol. 250, 527–552.

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