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

International Tables for Crystallography (2012). Vol. F, ch. 4.3, p. 135   | 1 | 2 |

Section 4.3.10. Improvement of crystal quality

Z. S. Derewendaa*

aDepartment of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908–0736, USA
Correspondence e-mail:

4.3.10. Improvement of crystal quality

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In most cases, protein engineering is used as a tool of last resort to obtain variants for proteins for which no crystals can be grown using the wild-type form. However, it may sometimes be necessary to obtain a new, different crystal form even when the wild-type protein does crystallize. Such a need may arise, for example, in drug-design investigations, where high-resolution structures are particularly critical for evaluation of the interactions between lead compounds and the target protein and may not always be possible using wild-type crystals. A novel crystal form may also be necessary if the wild-type crystals contain the target protein in an orientation in which the active site is obscured by crystal contacts, making it impossible to soak in drug lead compounds and screen small-molecule libraries by high-throughput crystallography (Blundell & Patel, 2004[link]).

One possible strategy for obtaining a new crystal form is to modify the existing crystal contacts by replacing some of the participating amino acids. While this approach occasionally leads to improvement of the X-ray data resolution (Liu et al., 2007[link]; Mizutani et al., 2008[link]), modification of crystal contacts is typically counterproductive as it abolishes the propensity of the target to crystallize in one form but does not necessarily induce another (Charron et al., 2002[link]). A more successful strategy is to generate a novel crystal form by engineering new crystal contacts through SER. For example, a novel crystal form of the insulin-like growth factor 1 receptor kinase domain, a putative drug target, was obtained using a double mutant (E1067A and E1069A); the new form diffracted to 1.5 Å resolution, whereas the wild-type crystals only diffracted to 2.7 Å resolution (Munshi et al., 2003[link]). In the case of the catalytic domain of activated factor XI, a key enzyme in the blood coagulation cascade and another potential drug target, a single K437A mutation allowed the preparation of a crystal form that diffracted to 2.0 Å resolution (Jin et al., 2005[link]). Entropy-reducing mutations were also key in the preparation of a crystal form of HIV-1 reverse transcriptase for structure-based drug design that diffracted to 1.8 Å resolution, in contrast to the typical 2.5–3.0 Å range observed for the wild-type protein crystals (Bauman et al., 2008[link]).


Bauman, J. D., Das, K., Ho, W. C., Baweja, M., Himmel, D. M., Clark, A. D. Jr, Oren, D. A., Boyer, P. L., Hughes, S. H., Shatkin, A. J. & Arnold, E. (2008). Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design. Nucleic Acids Res. 36, 5083–5092.
Blundell, T. L. & Patel, S. (2004). High-throughput X-ray crystallography for drug discovery. Curr. Opin. Pharmacol. 4, 490–496.
Charron, C., Kern, D. & Giegé, R. (2002). Crystal contacts engineering of aspartyl-tRNA synthetase from Thermus thermophilus: effects on crystallizability. Acta Cryst. D58, 1729–1733.
Jin, L., Pandey, P., Babine, R. E., Weaver, D. T., Abdel-Meguid, S. S. & Strickler, J. E. (2005). Mutation of surface residues to promote cryst­allization of activated factor XI as a complex with benzamidine: an essential step for the iterative structure-based design of factor XI inhibitors. Acta Cryst. D61, 1418–1425.
Liu, B., Luna, V. M., Chen, Y., Stout, C. D. & Fee, J. A. (2007). An unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba3 from Thermus thermophilus. Acta Cryst. F63, 1029–1034.
Mizutani, H., Saraboji, K., Malathy Sony, S. M., Ponnuswamy, M. N., Kumarevel, T., Krishna Swamy, B. S., Simanshu, D. K., Murthy, M. R. N. & Kunishima, N. (2008). Systematic study on crystal-contact engineering of diphthine synthase: influence of mutations at crystal-packing regions on X-ray diffraction quality. Acta Cryst. D64, 1020–1033.
Munshi, S., Hall, D. L., Kornienko, M., Darke, P. L. & Kuo, L. C. (2003). Structure of apo, unactivated insulin-like growth factor-1 receptor kinase at 1.5 Å resolution. Acta Cryst. D59, 1725–1730.

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