Effect of leucine-to-methionine substitutions on the diffraction quality of histone chaperone SET/TAF-Ibeta/INHAT crystals

Abstract

One of the most frequent problems in crystallization is poor quality of the crystals. In order to overcome this obstacle several methods have been utilized, including amino-acid substitutions of the target protein. Here, an example is presented of crystal-quality improvement by leucine-to-methionine substitutions. A variant protein with three amino-acid substitutions enabled improvement of the crystal quality of the histone chaperone SET/TAF-Ibeta/INHAT when combined with optimization of the cryoconditions. This procedure improved the resolution of the SET/TAF-Ibeta/INHAT crystals from around 5.5 to 2.3 A without changing the crystallization conditions.

Figure 1

Improvement of the TAF-IβΔC crystal by amino-acid substitution(s) and cryo-handling. R _mrgd-F values of the best data from each variant are plotted against resolution [(1/d)²]. DEF-a and DEF-b represent data from the crystals of variant DEF with and without optimization of the soaking conditions, respectively. These values were calculated using TRUNCATE in the CCP4 program suite (Collaborative Computational Project, Number 4, 1994 ▶).

Figure 2

Trehalose-binding geometry and sites of amino-acid substitutions. (a, b) The hydrogen-bond network around the bound trehalose at (a) binding site A (THL-A) and (b) binding site B (THL-B). Simulated-annealing OMIT maps for the bound trehaloses are also shown. The maps are contoured at the 3.5σ level. (c) The positions of bound trehalose at binding sites A and B (red) and of the introduced methionine residues, sites D, E and F (blue), are shown in the dimeric TAF-IβΔC molecule.