PDF(Pubmed)
Abstract：
The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.
摘要：
生物过程的快速动力学和相关的短寿命构象变化对在实时反应期间在结构上可视化生物分子的尝试提出了重大挑战。传统上,通过化学修饰或降低温度，提供有限的见解。这里,我们介绍了一种时间分辨低温EM方法，使用可重复使用的基于PDMS的微流控芯片组件，具有高反应物混合效率。用SiO2涂覆PDMS壁实际上消除了非特异性样品吸附，并确保了反应化学计量的维持，使其具有很高的可重复性。在10到1,000毫秒的工作范围内，该装置使我们能够跟踪生物分子的体外反应，其分辨率在3的范围内。通过使用这种方法，我们显示了在GTP存在下，通过在140ms内捕获三个高分辨率反应中间体，逐步HflX介导的70S大肠杆菌核糖体分裂的机制。
