申请人在前期工作中已成功解析了Picornavirus首个2C解旋酶结构"移除N端膜结合区的EV71-2C晶体结构"。我们发现，全长2C六聚体的组装是2C具有活性和EV71复制的必要条件；六聚体的形成依赖N-端膜结合区。为深入理解2C在EV71复制中的作用，我们拟研究全长2C六聚体的天然构象。为了克服2C不可溶问题，我们拟利用Nanodiscs技术，将全长2C六聚体植入磷脂双层上；利用冷冻电镜技术分别解析Nanodiscs-2C六聚体及其与ATP和RNA等形成复合物的高分辨结构。这些结构信息将揭示2C六聚体组装、亚基间协同完成ATP水解及解旋复制中间体-RNA双螺旋等的结构基础。本项目是前期工作的延续，基础扎实。用已知2C可溶片段的结构辅助完整六聚体电镜结构解析可显著降低技术难度。申请人具备多年肠道病毒复制酶结构与功能研究经验，技术平台成熟。依托单位已购置冷冻电镜，可支持本项目实施。

The applicant previously determined the first 2C helicase structure of Picornavirus"crystal structure of EV71-2C without N-terminal membrane binding region". We found that the assembly of full-length 2C hexamer is fundamental to 2C activity and EV71 replication; the N-terminal membrane binding region is important to hexamerization. In order to understand the role of 2C in EV71 replication, we will investigate nature conformation of full-length 2C hexamer. To overcome insolubility of 2C, we will use Nanodiscs technique to plant full-length 2C hexamer on phospholipid bilayer. We use cryo-EM technique to determine high-resolution structure of Nanodiscs-2C hexamer and the structures in complex with ATP and RNA substrate. These structural information will reveal structural basis for 2C hexamer assembling, the concerted work of multiple subunits in hydrolyzing ATP and the unwinding of replication intermediate (dsRNA). The current project is the continuation of our previous research on 2C. Using the crystal structure of the soluble EV71-2C fragment to assist the determination of Cryo-EM structure of full-length 2C hexamer can reduce the complexity of structure determination. The applicant has long-term experience on structural and functional studies of enterovirus replicases, the technical platform for structural and virological studies are well established. Our institute is equipped with electron microscope suitable for Cryo-EM study.