Abstract
Solving a novel RNA structure by x-ray crystallography requires a means to obtain initial phase estimates. This is a challenge because many of the tools available for solving protein structures are not available for RNA. We have developed a reliable means to use hexammine cations to address this challenge. The process involves engineering the RNA to introduce a reliable hexammine binding site into the structure, then soaking crystals of these RNAs with an iridium (III) or cobalt (III) compound in a "directed soaking" strategy. Diffraction data obtained from these crystals then can be used in SAD or MAD phasing. In many cases, suitable derivatives can be obtained by soaking the hexammine into RNA crystals that have not been engineered. Considerations for using this method and example protocols are presented.