MAD phasing using the (Ta₆Br₁₂)²⁺ cluster: a retrospective study

The crystal structure of cytokinin-specific binding protein (CSBP) containing four independent molecules with 4 × 155 = 620 residues in the asymmetric unit of the P6₄ unit cell has been solved by three-wavelength MAD using 1.8 Å resolution data recorded from a crystal derivatized with the dodeca­bromohexatantalum cation (Ta₆Br₁₂)²⁺. The diffraction data contained a very strong anomalous signal (allowing successful phasing even using peak SAD data alone) despite the fact that the five (Ta₆Br₁₂)²⁺ clusters found in the asymmetric unit have low occupancy (about 0.3). The derivative structure has been successfully refined to R = 0.158, providing interesting details on the geometry of the (Ta₆Br₁₂)²⁺ cluster, its interactions with the protein and on the backsoaking of a cytokinin ligand that was originally part of a CSBP-cytokinin complex in the native crystals used for (Ta₆Br₁₂)²⁺ derivatization. A simulation analysis of the phasing power of the (Ta₆Br₁₂)²⁺ ions at artificially imposed resolution limits shows that it is not possible to resolve the individual Ta atoms if the d_min limit of the data is higher than 2.9 Å. Additionally, for successful Ta identification the (Ta₆Br₁₂)²⁺ complex should be specifically bound and ordered. Good binding at the protein surface is facilitated by the presence of acidic groups, indicating higher pH buffer conditions to be preferable. In addition, the water channels in the crystal should be sufficiently wide (at least 11 Å) to allow free diffusion of the (Ta₆Br₁₂)²⁺ ions on soaking. A retrospective look at the initial molecular-replacement calculations provides interesting insights into how the peculiar packing mode and strong bias of the molecular-replacement-phased electron-density maps had hindered successful solution of the structure by this method.