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Author:
Date:
2015-09-05
[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 ...
[摘要] 与最近在复杂的非编码RNA的基因组范围和功能研究的数量的显着增长相比，机械和结构分析已经落后。在大RNA及其复合物的结构确定中的主要技术瓶颈是衍射晶体的制备。经验上，由于其固有的无序和在晶体内的非特异性堆积，绝大多数这样的RNA晶体不能将X射线衍射到可用的分辨率（〜4）。在这里，我们提出一个协议，结合后结晶阳离子替代和脱水，大大改善晶体的大基因调节mRNA-tRNA复杂的衍射质量。该程序不仅将X射线数据的分辨率极限从8.5扩展到3.2，而且还显着提高了数据的质量，实现了重新定相和结构确定。因为它利用了反离子和溶剂化在RNA结构中的一般重要性，这个程序可能证明在其他大型非编码RNA的晶体学分析中广泛有用。
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