A general module for RNA crystallization
A general module for RNA crystallization
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DOI：
10.1006/jmbi.1998.1789
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发表时间：
1998-06-12
影响因子：
5.6
通讯作者：
Doudna, JA
中科院分区：
文献类型：
--
作者：
Ferré-D'Amaré, AR;Zhou, KH;Doudna, JA
除了简单的寡核苷酸双链体之外，RNA分子的结晶仍然是X射线晶体学结构测定中具有挑战性的步骤。使生物化学、共价和构象均质的目标分子经受详尽的结晶条件通常不足以产生足够大的晶体以用于X射线数据收集。即使获得了大的RNA晶体，它们通常也不能将X射线转换到可以产生生物化学信息结构的分辨率。我们推断，折叠良好的RNA分子通常会呈现出由磷酸骨架主导的基本未分化的分子表面。在晶体成核和生长过程中，这可能导致相邻分子巧妙地堆积而不对齐，导致过早的晶体生长停止和无序。为了克服这个问题，我们开发了一种结晶模块，该模块由正常的分子内RNA-RNA相互作用组成，该相互作用被招募以进行分子间晶体接触。靶RNA分子被改造成在不影响生物化学活性的位点含有该模块。结晶模块的存在似乎驱动晶体生长，在此过程中形成其他非设计的接触。我们已经成功地采用GAAA四环/四环受体相互作用来结晶许多II组内含子结构域5-结构域6和丁型肝炎病毒（HDV）核酶RNA构建体。该模块的使用允许大晶体的容易生长，使得筛选大量晶体形式以获得有利的衍射特性变得实用。该方法已经导致II组内含子结构域晶体，其将X射线辐射抑制到3.5埃分辨率。(C)出版社：Academic Press Limited。
Crystallization of RNA molecules other than simple oligonucleotide duplexes remains a challenging step in structure determination by X-ray crystallography. Subjecting biochemically, covalently and conformationally homogeneous target molecules to an exhaustive array of crystallization conditions is often insufficient to yield crystals large enough for X-ray data collection. Even when large RNA crystals are obtained, they often do not diffract X-rays to resolutions that would lead to biochemically informative structures. We reasoned that a well-folded RNA molecule would typically present a largely undifferentiated molecular surface dominated by the phosphate backbone. During crystal nucleation and growth, this might result in neighboring molecules packing subtly out of register, leading to premature crystal growth cessation and disorder. To overcome this problem, we have developed a crystallization module consisting of a normally intramolecular RNA-RNA interaction that is recruited to make an intermolecular crystal contact. The target RNA molecule is engineered to contain this module at sites that do not affect biochemical activity. The presence of the crystallization module appears to drive crystal growth, in the course of which other, non-designed contacts are made. We have employed the GAAA tetraloop/tetraloop receptor interaction successfully to crystallize numerous group II intron domain 5-domain 6, and hepatitis delta virus (HDV) ribozyme RNA constructs. The use of the module allows facile growth of large crystals, making it practical to screen a large number of crystal forms for favorable diffraction properties. The method has led to group II intron domain crystals that diffract X-radiation to 3.5 Angstrom resolution. (C) 1998 Academic Press Limited.