Procedures that were critical for crystallization of theglmSribozyme-riboswitch RNA domain from the thermophilic Gram-positive bacteriumThermoanaerobacter tengcongensisare described. Experimental design based on screening multiple variant RNA sequences and techniques used to identify initial crystallization conditions were similar to those employed for most RNAs. However, serendipitous in-drop digestion of one RNA construct at a specific internucleotide linkage was crucial for the growth of high-qualityglmSribozyme crystals. Biochemical analysis of crystalline RNA identified the site of scission and guided design of an optimized bimolecular RNA construct. Finally, modifications of ionic strength and pH of solutions used for stabilization of the crystals were essential for optimal diffraction and binding of the activator glucosamine-6-phosphate, respectively. Although their details are specific to theglmSribozyme, these general strategies may be useful for analyzing and improving crystals of other RNAs.
