Although we were able to obtain cube-shaped crystals of XC1936 (Fig. 2
) using the original purification buffer, we found that their diffraction quality was not good enough to solve the required protein phase angles. We used two additional steps to obtain crystals that were suitable for the determination of the X. campestris
UMPK structure in the apo form. The first step involved selecting a better buffer than that used during purification in order to increase the solubility and decrease the aggregation of XC1936. Initially, we used the screening buffers defined by Jancarik et al.
). However, soon after we found that buffers with different pH values may exhibit very different effects on the protein solubility. Hence, we developed an expanded set of screen buffers with finer pH steps as shown in Table 1. Several extra salts such as sodium formate, sodium sulfate, lithium chloride, magnesium chloride, calcium chloride, potassium chloride and ammonium chloride were included as it has been reported that these salts can further increase protein solubility (Izaac et al.
). In addition, we also included two extra nonionic surfactants (0.1% Tween-20 and 0.1% Triton X-100) to expand the additive screen (Chou et al.
). However, using the optimum buffer (20 mM
ADA pH 6.8, 5 mM
β-mercaptoethanol and 0.02% NaN3
) obtained from this screening step, the resulting XC1936 crystals still did not seem to be greatly improved in terms of either their appearance (the crystal surface appeared rugged; Fig. 2
) or their diffraction quality.
It was reasoned that the precipitation rate of the XC1936 protein may have been too fast and that the application of a strong superconducting magnetic field may partially reduce gravity and hence the sedimentation speed (Lin et al.
). When XC1936 was dissolved in the same optimum buffer and crystallization was performed in the same manner, but with the crystallization plates placed alongside a strong 18.7 T superconducting magnet, it was observed that the crystal quality indeed improved significantly, as judged by the sharper edges of the crystals and the improved diffraction resolution (Table 2). The average dimensions of these crystals were 0.13 × 0.1 × 0.1 mm after 7 d and the crystals were highly suitable for structural determination.
Importantly, preliminary structural analysis of the apo-form XC1936 hexamer revealed a well determined electron-density map in the loop (56NIFRGAGLAAS66) partially responsible for substrate/substrate-analogue binding (data to be published), indicating that application of a strong magnetic field can be very beneficial in determination of the structure of flexible loops. As we have also successfully obtained cocrystals of XC1936 UMPK with several of its cofactors, such as UMP, UDP, UTP, AMPPNP and GTP (data not shown), detailed structural comparison between apo-form UMPK from X. campestris and its substrate/substrate analogue/cofactor complexes could reveal interesting conformational changes associated with ligand binding. We are currently collecting further experimental data and detailed structural studies are now under way.