Ke Ailong, Doudna Jennifer A
Department of Molecular and Cell Biology and Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
Methods. 2004 Nov;34(3):408-14. doi: 10.1016/j.ymeth.2004.03.027.
RNA plays a direct role in a variety of cellular activities, and in many cases its biological function is conferred by the RNA three-dimensional structure. X-ray crystallography is the method of choice for determining high resolution structures of large RNA molecules, and can also be used to compare related RNAs and identify conformational changes that may accompany biochemical activity. However, crystallization remains the rate-limiting step in RNA structure determination due to the difficulty in obtaining well-ordered crystals for X-ray diffraction analysis. Several approaches to sample preparation, crystallization, and crystal handling are presented that have been used successfully in the structure determination of RNA and RNA-protein complexes in our laboratory, and should be generally applicable to RNAs in other systems.
RNA在多种细胞活动中发挥直接作用，并且在许多情况下，其生物学功能由RNA三维结构赋予。X射线晶体学是确定大型RNA分子高分辨率结构的首选方法，也可用于比较相关RNA并识别可能伴随生化活性的构象变化。然而，由于难以获得用于X射线衍射分析的有序晶体，结晶仍然是RNA结构测定中的限速步骤。本文介绍了几种样品制备、结晶和晶体处理方法，这些方法在我们实验室中已成功用于RNA和RNA-蛋白质复合物的结构测定，并且通常适用于其他系统中的RNA。
