Developing fast cryo-EM sample preparation for time-resolved structural studies

Single particle cryo-electron microscopy (cryo-EM) has become one of the major techniques for protein structure determination, through advances in microscope hardware and image processing software. The resolution of cryo-EM and the speed of data collection are approaching those of X-ray crystallography, in addition cryo-EM allows determination of more dynamic and flexible structures. While computational methods that account for dynamics are being developed, the vast majority of cryo-EM structures is currently determined under equilibrium conditions. In order to fulfil their functions, proteins partake in reactions and are usually in environments far from equilibrium. To determine the structural dynamics of proteins in non-equilibrium systems, a series of structural snapshots can be recorded over time after triggering the reaction of interest. In principle, this allows a ‘molecular movie’ to be assembled, which promises a much improved understanding of the relationship between protein structure, dynamics and function. However, recording such ‘molecular movies’ is experimentally challenging and requires the use of time-resolved structural techniques, such as time-resolved cryo-EM. In time-resolved cryo-EM (TrEM) a reaction is initiated, often by rapid mixing, and quenched upon vitrification. The majority of biological reactions is faster than conventional cryo-EM grid preparation, so TrEM requires specialised instrumentation. In this thesis, methods for TrEM are presented, characterised and developed further. Different approaches for fast sample application and rapid mixing are compared and the effect of sample exposure to the air-water interface is analysed. Together, the thesis contributes to a better understanding of fast grid preparation for TrEM and aims to give directions for future TrEM studies. Using the methods established in this thesis, the structure of the pre-power stroke actomyosin complex is solved, demonstrating the power of TrEM and providing insight into the molecular mechanism of force generation by the actomyosin system.