Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado BioFrontiers Institute, Boulder, CO 80309-0596, USA.
Structure. 2018 Sep 4;26(9):1275-1283.e3. doi: 10.1016/j.str.2018.05.005. Epub 2018 Jun 14.
Crystallography is a major technique for determining large RNA structures. Obtaining diffraction-quality crystals has been the bottleneck. Although several RNA crystallization methods have been developed, the field strongly needs additional approaches. Here we invented an in crystallo selection strategy for identifying mutations that enhance a target RNA's crystallizability. The strategy includes constructing an RNA pool containing random mutations, obtaining crystals, and amplifying the sequences enriched by crystallization. We demonstrated a proof-of-principle application to the P4-P6 domain from the Tetrahymena ribozyme. We further determined the structures of four selected mutants. All four establish new crystal lattice contacts while maintaining the native structure. Three mutants achieve this by relocating bulges and one by making a helix more flexible. In crystallo selection provides opportunities to improve crystals of RNAs or RNA-ligand complexes. Our results also suggest that mutants may be rationally designed for crystallization by "walking" a bulge along the RNA chain.
结晶学是确定大型 RNA 结构的主要技术。获得具有衍射质量的晶体一直是瓶颈。尽管已经开发了几种 RNA 结晶方法，但该领域非常需要其他方法。在这里，我们发明了一种在晶体中选择的策略，用于鉴定增强靶 RNA 结晶能力的突变。该策略包括构建一个包含随机突变的 RNA 池，获得晶体，并扩增晶体中富集的序列。我们用四膜虫核酶的 P4-P6 结构域证明了一个原理验证应用。我们进一步确定了四个选定突变体的结构。这四个突变体都建立了新的晶格接触，同时保持了天然结构。三个突变体通过重新定位凸起来实现这一点，而一个通过使螺旋更加灵活来实现。晶体中选择为改善 RNA 或 RNA-配体复合物的晶体提供了机会。我们的结果还表明，通过沿着 RNA 链“行走”凸起，突变体可以合理地设计用于结晶。
