The structure determination of complex RNA molecules such as ribozymes, riboswitches and aptamers by X-ray crystallography hinges on the preparation of well-ordered crystals. Success usually results from molecular engineering to facilitate crystallization. An approach that has resulted in 10 new RNA structures in the past decade is the use of the U1A crystallization module. In this approach, the cognate site for the U1A spliceosomal protein is introduced into a functionally dispensable location in the RNA of interest, and the RNA is cocrystallized with the basic RNA-binding protein. In addition to facilitating crystallization, the presence of U1A can be useful for de novo phase determination. In this paper, some general considerations for the use of this approach to RNA crystallization are presented, and specifics of the application of the U1A module to the crystallization of the hairpin ribozyme and the tetracycline aptamer are reviewed.
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