Determining structures of RNA aptamers and riboswitches by X-ray crystallography (original) (raw)
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X-ray crystallography remains a powerful method to gain atomistic insights into the catalytic and regulatory functions of RNA molecules. However, the technique requires the preparation of diffraction-quality crystals. This is often a resource- and time-consuming venture because RNA crystallization is hindered by the conformational heterogeneity of RNA, as well as the limited opportunities for stereospecific intermolecular interactions between RNA molecules. The limited success at crystallization explains in part the smaller number of RNA-only structures in the Protein Data Bank. Several approaches have been developed to aid the formation of well-ordered RNA crystals. The majority of these are construct-engineering techniques that aim to introduce crystal contacts to favor the formation of well-diffracting crystals. A typical example is the insertion of tetraloop–tetraloop receptor pairs into non-essential RNA segments to promote intermolecular association. Other methods of promoting...
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The crystallization and structural determination of large RNAs and their complexes remain major bottlenecks in the mechanistic analysis of cellular and viral RNAs. Here, we describe a protocol that combines post-crystallization dehydration and ion replacement that dramatically improved the diffraction quality of crystals of a large gene-regulatory tRNA-mRNA complex. Through this method, the resolution limit of X-ray data extended from 8.5 to 3.2 Å, enabling structure determination. Although this protocol was developed for a particular RNA complex, the general importance of solvent and counterions in nucleic acid structure may render it generally useful for crystallographic analysis of other RNAs.