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

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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.