Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.
Methods. 2010 Oct;52(2):168-72. doi: 10.1016/j.ymeth.2010.06.011. Epub 2010 Jun 9.
The crystallographic phase problem is the primary bottleneck encountered when attempting to solve macromolecular structures for which no close crystallographic structural homologues are known. Typically, isomorphous "heavy-atom" replacement and/or anomalous dispersion methods must be used in such cases to obtain experimentally-determined phases. Even three-dimensional NMR structures of the same macromolecule are often not sufficient to solve the crystallographic phase problem. RNA crystal structures present additional challenges due to greater difficulty in obtaining suitable heavy-atom derivatives. We present a unique approach to solve the phase problem for novel RNA crystal structures that has enjoyed a reasonable degree of success. This approach involves modeling only those portions of the RNA sequence whose structure can be predicted readily, i.e., the individual A-form helical regions and well-known stem-loop sub-structures. We have found that no prior knowledge of how the helices and other structural elements are arranged with respect to one another in three-dimensional space, or in some cases, even the sequence, is required to obtain a useable solution to the phase problem, using simultaneous molecular replacement of a set of generic helical RNA fragments.
晶体学相问题是尝试解决未知近晶学结构同源物的大分子结构时遇到的主要瓶颈。通常情况下，在这种情况下必须使用同晶“重原子”置换和/或反常散射方法来获得实验确定的相。即使是同一大分子的三维 NMR 结构通常也不足以解决晶体学相问题。由于获得合适的重原子衍生物更加困难，RNA 晶体结构带来了额外的挑战。我们提出了一种独特的方法来解决新 RNA 晶体结构的相问题，该方法取得了相当程度的成功。这种方法仅涉及对那些结构可以轻易预测的 RNA 序列部分进行建模，即单个 A 型螺旋区域和众所周知的茎环亚结构。我们发现，在某些情况下，甚至不需要知道螺旋和其他结构元件在三维空间中的排列方式，或者序列，就可以使用一组通用的螺旋 RNA 片段同时进行分子置换，从而获得相问题的可用解决方案。
