Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase

Myllykoski, Matti; Kursula, Petri

For crystallization, LigT was in the gel filtration buffer at 10 mg/ml. Sitting drop crystallization experiments were prepared with 2:1, 1:1, and 1:2 drop ratios. The crystallization conditions were composed of 0.1 M Tris-HCl at pH 7.4-7.5, 0.2 M MgCl 2 , and PEG 8000 at 12-20% (w/ v). For obtaining liganded complexes, LigT was mixed with putative active-site ligands prior to crystallization at 5 mM ligand concentration; these compounds included ATP, NADP + , 2 0 ,3 0 -cAMP, and 3 0 -AMP. Crystals were cryoprotected by soaking them for a few minutes in the well solution supplemented with the ligand (if present) and 15% (v/v) PEG 200. X-ray diffraction data were collected using synchrotron radiation at 100 K. Data were collected on beamlines P11 and P13 [37] at PETRA III/DESY (Hamburg, Germany), as well as on beamline I911-2 at MAX-Lab (Lund, Sweden). All data were processed using XDS [38] .