Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method

Abstract

A tetrahydrofolate-dependent O-demethylase, LigM, from Sphingobium sp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P3₁21 or P3₂21 crystals, which diffracted to 2.5-3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P3₁21/P3₂21 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5-3.0 Å resolution and belonged to space group P2₁2₁2, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P2₁2₁2 crystals diffracted to better than 2.0 Å resolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.

Keywords: O-demethylase; Sphingobium sp. SYK-6; cryoprotectant; hemihedral twinning; microseeding; tetrahydrofolate.

Figure 1

Generation of one-carbon THF by O-demethylation of LMW lignins (a) and by the glycine-cleavage system (b).

Figure 2

P2₁2₁2 crystals of LigM. The largest dimension of a crystal is approximately 500 µm.

Figure 3

X-ray diffraction pattern of an LigM crystal. The diffraction data were collected on BL17A of PF using a PILATUS3 S6M detector. The dotted circle corresponds to a resolution of 2.0 Å.