Edwards Andrea L, Garst Andrew D, Batey Robert T
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA.
Methods Mol Biol. 2009;535:135-63. doi: 10.1007/978-1-59745-557-2_9.
Structural biology plays a central role in gaining a full understanding of the myriad roles of RNA in biology. In recent years, innovative approaches in RNA purification and crystallographic methods have lead to the visualization of an increasing number of unique structures, providing new insights into its function at the atomic level. This article presents general protocols which have streamlined the process of obtaining a homogeneous sample of properly folded and active RNA in high concentrations that crystallizes well in the presence of a suitable heavy-atom for phasing. Of particular importance are approaches toward RNA crystallography that include exploring "construct space" as opposed to "condition space". Moreover, development of a highly flexible method for experimentally phasing RNA crystals may open the door to a relatively simple means of solving these structures.
结构生物学在全面理解RNA在生物学中的众多作用方面发挥着核心作用。近年来，RNA纯化和晶体学方法的创新途径已导致越来越多独特结构的可视化，为其在原子水平上的功能提供了新的见解。本文介绍了一些通用方案，这些方案简化了获得高浓度、折叠正确且有活性的RNA均匀样品的过程，该样品在存在合适的重原子用于相位测定时能很好地结晶。特别重要的是RNA晶体学方法，包括探索“构建空间”而非“条件空间”。此外，开发一种用于实验性确定RNA晶体相位的高度灵活方法可能为解决这些结构提供一种相对简单的手段打开大门。
