Keel Amanda Y, Rambo Robert P, Batey Robert T, Kieft Jeffrey S
Department of Biochemistry and Molecular Genetics, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.
Structure. 2007 Jul;15(7):761-72. doi: 10.1016/j.str.2007.06.003.
X-ray crystallography of biologically important RNA molecules has been hampered by technical challenges, including finding heavy-atom derivatives to obtain high-quality experimental phase information. Existing techniques have drawbacks, limiting the rate at which important new structures are solved. To address this, we have developed a reliable means to localize heavy atoms specifically to virtually any RNA. By solving the crystal structures of thirteen variants of the G*U wobble pair cation binding motif, we have identified a version that when inserted into an RNA helix introduces a high-occupancy cation binding site suitable for phasing. This "directed soaking" strategy can be integrated fully into existing RNA crystallography methods, potentially increasing the rate at which important structures are solved and facilitating routine solving of structures using Cu-Kalpha radiation. This method already has been used to solve several crystal structures.
生物重要RNA分子的X射线晶体学一直受到技术挑战的阻碍，包括寻找重原子衍生物以获得高质量的实验相位信息。现有技术存在缺陷，限制了重要新结构的解析速度。为解决这一问题，我们开发了一种可靠的方法，可将重原子特异性地定位到几乎任何RNA上。通过解析G*U摆动对阳离子结合基序的13种变体的晶体结构，我们确定了一个版本，当将其插入RNA螺旋中时，会引入一个适合相位测定的高占有率阳离子结合位点。这种“定向浸泡”策略可以完全整合到现有的RNA晶体学方法中，有可能提高重要结构的解析速度，并有助于使用Cu-Kα辐射常规解析结构。该方法已被用于解析多个晶体结构。
