Abstract
Compared to the recent dramatic growth in the numbers of genome-wide and functional studies of complex non-coding RNAs, mechanistic and structural analyses have lagged behind. A major technical bottleneck in the structural determination of large RNAs and their complexes is preparation of diffracting crystals. Empirically, a vast majority of such RNA crystals fail to diffract X-rays to usable resolution (~4 Å) due to their inherent disorder and non-specific packing within the crystals. Here, we present a protocol that combines post-crystallization cation replacement and dehydration that dramatically improved the diffraction quality of crystals of a large gene-regulatory mRNA-tRNA complex. This procedure not only extended the resolution limit of X-ray data from 8.5 to 3.2 Å, but also significantly improved the quality of the data, enabling de novo phasing and structure determination. Because it exploits the general importance of counterions and solvation in RNA structure, this procedure may prove broadly useful in the crystallographic analyses of other large non-coding RNAs.