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, pp. 101-102   | 1 | 2 |

Section 4.3.5. Mutations to improve diffraction quality

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:

4.3.5. Mutations to improve diffraction quality

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Another commonly encountered situation is that crystals can be obtained, but they diffract poorly. There are many examples where investigators have applied protein engineering in an effort to overcome this problem.

Proteolytic trimming is one possible approach to improving diffraction quality. For example, Zhang et al. (1997[link]) attempted to crystallize a homodimer of the C2 domain of adenylyl cyclase. The initial crystals diffracted poorly (to 3.8 Å), so the effects of limited proteolysis with chymotrypsin, trypsin, GluC and LysC were investigated. A stable cleavage product was observed with GluC, approximately 4 kDa smaller than the full-length protein, but in order to avoid minor products formed during GluC proteolysis, the cleavage site was re-engineered as a thrombin site. Since there was already an atypical thrombin site seven residues from this site, proteolysis resulted in a smaller protein than expected; nevertheless, this modified protein crystallized readily and diffracted to 2.2 Å.

The importance of applying a variety of strategies to improve crystal quality is exemplified by the work of Oubridge et al. (1995[link]), in which initial attempts to crystallize wild-type U1A complexed with RNA hairpins resulted in cubic crystals diffracting to 7–8 Å. By mutating surface residues, changing the N-terminal sequence to reduce heterogeneity and varying the sequence of the RNA hairpin, a new crystal form which diffracted to 1.7 Å was ultimately crystallized. However, in order to achieve this result, many variants were constructed and examined. For the protein, mutations were introduced which it was believed (incorrectly) would affect the crystal packing, and which were selected based on the observed similarity of space group and cell dimensions between crystals of the complex and those of the protein alone. One of these mutations, together with an additional mutation resulting from a polymerase chain reaction (PCR) artefact, yielded crystals that diffracted to 3.5 Å. Additional variation of the length and composition of the RNA hairpin led to a new crystal form of this double mutant in the presence of a 21-base RNA that diffracted to 1.7 Å. A further mutation, [\hbox{Ser29}\rightarrow \hbox{Cys}], was made to allow mercury binding (see Section 4.3.8[link]), also resulting in crystals that diffracted to 1.7 Å. The authors commented that `If any principle emerges from this study, it is that the key to success is not in concentrating on exhausting any one approach, but in the diversity of approaches used.'

The relevance of this comment is illustrated by the attempts of Scott et al. (1998)[link] to obtain diffraction-quality crystals of the I-[\hbox{A}^{\rm d}] class II major histocompatibility complex (MHC) protein. This complex exists in vivo as a heterodimer, but expression in recombinant form did not lead to satisfactory dimer formation. A leucine zipper peptide was therefore added to each chain to enhance dimerization. Attempts to crystallize this heterodimer after removal of the leucine zippers and in the presence of bound peptides led to poorly diffracting crystals. To enhance the affinity of an ovalbumin peptide for the MHC dimer, the peptide was then attached through a six-residue linker to the N-terminus of the chain, tethering it in the vicinity of the binding site. This construct, in conjunction with removal of the leucine zippers from the heterodimer, resulted in crystals that diffracted to 2.6 Å.


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.Google Scholar
Scott, C. A., Garcia, K. C., Stura, E. A., Peterson, P. A., Wilson, I. A. & Teyton, L. (1998). Engineering protein for X-ray crystallography: the murine major histocompatibility complex class II molecule I-A. Protein Sci. 7, 413–418.Google Scholar
Zhang, G., Liu, Y., Qin, J., Vo, B., Tang, W.-J., Ruoho, A. E. & Hurley, J. H. (1997). Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase. Protein Sci. 6, 903–908.Google Scholar

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