Time-resolved methodologies to provide both spatial and temporal resolution in Electron Microscopy

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Structural biology has been key to unlocking the secrets of mutational change within proteins and provides the platform for therapeutic intervention. Traditionally, X-ray crystallography has provided the high resolution information required, but it is dependent on well-ordered crystals, limiting its scope, especially for membrane proteins and large protein complexes. Yet these systems demand structural investigation. Alternatively, electron microscopy (EM) is ideally suited to studying protein complexes and membrane proteins but has traditionally been hindered by a more modest resolution. However, EM has undergone a "resolution revolution" driven by more stable microscopes, advanced processing algorithms, rapid automatic data collection and new direct electron detectors which offer greater quality images. As a consequence atomic resolution of non-symmetric proteins is becoming the norm and previously intractable but important medical targets are now amenable to structure based drug design using EM derived structures. A powerful but under exploited advantage of EM is the ability to trap different conformational states that exist within the sample. However, this is currently reliant on computational sorting of different conformational states that exist within the sample in the "ground state". To overcome this, time-resolved methodologies can be used. This methodology relies on mixing a protein and substrate rapidly before freezing in a liquid cryogen to trap the reaction intermediate in the ms time frame. The significant challenge is that to image a protein with the electron microscope it must be frozen on a specialised grid in vitreous ice which is thin enough for the electrons to pass through but not thinner than the sample being studied. This is commonly achieved using an automated "blotting" device (such as the Vitrobot), which blots away excess solution from the grid leaving a suitable amount such that upon rapid freezing a vitreous ice layer is formed of an appropriate thickness. Our primary route for time-resolved studies will be to rapidly mix protein and substrate in a microfluidic chamber before directly spraying it onto a grid and plunge freezing. We have a basic setup that is capable of a time resolution >50ms but methodologies are still in their infancy and have yet to realise their full potential. This is limited by two major factors, the first is in obtaining a suitable ice thickness with inconsistencies of the direct spraying approach. By screening a range of grid types including the recently developed micro-fibre grids that have been designed to negate this problem we are confident we can find a working solution. The second limitation is in obtaining a reliable, reproducible and fast rapid mixing device. Through a new collaboration with Hamburg University we will translate the technology designed for cutting edge diffraction technologies (x-ray free electron laser and SAXS) to develop a rapid mixing unit. By integrating with the leading grid preparation system (Vitrobot) we can develop a universal system accessible by any interested EM group, moving this technology from niche to mainstream. This funding will allow us to take the first significant step towards this by generating a prototype system capable of producing time resolution >5ms in a more controlled and reliable manner. Looking forward the next significant milestone in structural biology will be the capture of temporal as well as spatial resolution, greatly increasing our understanding of proteins and protein complexes and moving us away from the "static" view that currently predominates in the field.