ADP-2Ho as a phasing tool for nucleotide-containing proteins

Trivalent holmium ions were shown to isomorphously replace magnesium ions to form an ADP-2Ho complex in the nucleotide-binding domain of Bacillus subtilis 5-methylthioribose (MTR) kinase. This nucleotide–holmium complex provided sufficient phasing power to allow SAD and SIRAS phasing of this previously unknown structure using the L_III absorption edge of holmium. The structure of ADP-2Ho reveals that the two Ho ions are approximately 4 Å apart and are likely to share their ligands: the phosphoryl O atoms of ADP and a water molecule. The structure determination of MTR kinase using data collected using Cu Kα X-radiation was also attempted. Although the heavy-atom substructure determination was successful, interpretation of the map was more challenging. The isomorphous substitution of holmium for magnesium in the MTR kinase–nucleotide complex suggests that this could be a useful phasing tool for other metal-dependent nucleotide-containing proteins.