Developing new approaches and improved technologies for the study of membrane protein structure and mechanism

背景：在过去的十年中，随着显微镜硬件和图像处理的显著改进，CryoEM经历了一场“分辨率革命”。这场革命的第二波正在进行中，电子显微镜的样品制备正在快速发展，新的方法可以提高重复性，并通过时间分辨方法捕获短暂的中间步骤。更好地理解管理样品制备的一些基本规则将使我们能够看到更具挑战性的蛋白质系统。此外，时间分辨方法学的发展将使IS能够探索蛋白质和蛋白质复合体的机制，特别是膜蛋白质的机制。该项目是利兹大学和SPT LabTech的合作项目，将调查蛋白质大小、浓度和组成对网格冰层内行为的作用。然后，我们将使用快速混合来观察药物外排和通道形成等过程。目标：1.使用已建立的工作流程调查膜蛋白大小、组成和浓度对结果网格的影响。2.进一步发展快速混合方法，以及多个样本对结果网格质量的影响。使用改进后的方案以时间依赖的方式研究一系列生物系统。我们希望这项工作将使我们能够提供更有效和更可重复的样品制备方法，同时也为关键的生物过程提供新的线索。方法论和基础科学的结合将提供令人兴奋的新见解，可能会对该领域的其他人产生广泛影响。

Background: CryoEM has undergone a 'resolution revolution' in the last decade, with dramatic improvements in microscope hardware and image processing. A second wave of this revolution is now underway, with sample preparation for electron microscopy under rapid development, with new methodologies to improve reproducibility and trap short lived intermediate steps by time-resolved methods. A better understanding of some of the basic rules that govern sample preparation will allow us to look at more challenging protein systems. Moreover, development of time-resolved methodologies will allow is to explore the mechanism of proteins and protein complexes, especially for membrane proteins. This project is a collaboration between the University of Leeds and SPT Labtech and will investigate the role of protein size, concentration and composition on behaviour within the grid ice layer. We will then use fast mixing to look at processes such as drug efflux and channel formation.Objectives:1. Investigate the effects of membrane protein size, composition and concentration on the resultant grid using established workflows. 2. Further develop the rapid mixing approach and how multiple specimens affect the resultant grid quality.3. Use the improved protocols to study a range of biological systems in a time dependant manner.We hope that this work will allow us to provide more efficient and reproducible means of sample preparation whilst also shedding new light on key biological processes. The mixture of methodology and fundamental science will provide exciting new insights which could have a broad impact on others in the field.