Department of Chemistry, University of Chicago, Chicago, Illinois, USA.
Nat Struct Mol Biol. 2011 Jan;18(1):100-6. doi: 10.1038/nsmb.1945. Epub 2010 Dec 12.
RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab-ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs' high molecular weight, large surface area and phasing power.
RNA 结晶和相分析是 RNA 结构测定的主要瓶颈。为了利用抗体片段作为 RNA 结晶辅助因子，我们使用富含精氨酸的合成 Fab 噬菌体展示文库来获得针对 I 类连接酶核酶的 Fab。我们使用 Fab 坐标的分子置换法解析了一个 3.1-Å 分辨率的 Fab-连接酶复合物结构，证实了核酶的结构，并揭示了该辅助因子在 RNA 识别和晶体接触中的作用。表位位于帽状 P5 螺旋的 GAAACAC 序列中，并且该序列在其他结构 RNA 的背景下保留了高亲和力 Fab 结合。这种可移植的表位提供了一个新的 RNA 结晶辅助因子系统，它可以与 U1A RNA 结合蛋白平行筛选，具有较小的环和 Fab 的高分子量、大表面积和相分析能力的优势。
