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, pp. 133-134   | 1 | 2 |

Section 4.3.7. Removal of post-translational modifications

Z. S. Derewendaa*

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

4.3.7. Removal of post-translational modifications

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A number of proteins undergo post-translational modifications which can adversely affect crystallization. By far the most ubiquitous is N- and O-glycosylation, primarily of membrane-associated, secreted and lysosomal proteins. In a number of cases successful crystallization of glycoproteins purified from natural sources has been reported and carbohydrate groups have often been found to be ordered and occasionally sequestered between the protein molecules, thus even contributing in a positive way to crystallization (Mark et al., 2003[link]; Aleshin et al., 1994[link]). In general terms, however, the flexible and heterogeneous carbohydrate moieties, particularly the oligosaccharides linked by N-glycosyl­ation, can account for a significant fraction of the surface area of the protein and can therefore be detrimental to crystallization. The preparation of recombinant proteins in E. coli eliminates these post-translational modifications and may sometimes solve the problem (Mohanty et al., 2009[link]), but N-glycosylation is often required for appropriate folding and solubility, so this approach is not always possible. However, if a eukaryotic expression system is a necessity, the problem can often be resolved by mutating the asparagines within the relevant glycosylation motifs (Asn-X-Thr/Ser), e.g. to aspartates, as was performed in the case of the extracellular domain of the metabotropic glutamate receptor expressed in insect cells (Muto et al., 2009[link]), or to glutamines, as was performed for the human testis angiotensin-converting enzyme (Gordon et al., 2003[link]). Alternatively, glycosylation at these sites can be eliminated by mutation of the Thr/Ser residues in the glycosylation motif to alanine or other amino acids, as described for rat cathepsin B (Lee et al., 1990[link]), or valine, as was the case with the Ebola virus glycoprotein (Lee et al., 2008[link], 2009[link]). Similarly, potentially glycosylated threonines or serines in O-glycosyl­ated glycoproteins can be mutated to other amino acids (Horan et al., 1998[link]) to avoid or reduce glycosylation.

Other post-translational modifications occur less frequently. Prenylation and N-myristoylation can occur at the C- and N-­termini, respectively. Expression in E. coli, often using truncated versions of target proteins, is a common remedy (Pai et al., 1990[link]).


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