Understanding the rules of sample preparation for single particle cryo-EM

Structural biology is an important area of science. By understanding the structure of a protein or protein complexes we can start to understand how it works and how mutations cause disease. Furthermore, by understanding a proteins structure we can start to design new therapeutics that can modulate its function to treat different disease states. However, obtaining a structure is not easy, proteins are very small, dynamic and delicate. One of the main techniques which has grown significantly in recent years is cryo electron microscopy cryo-EM which allows you to see proteins at atomic resolution where we can see the position of individual atoms. Whilst the microscopes and the way in which we process the data are well developed there is a current limitation in our understanding of how we prepare the sample for the microscope. This preparation involves freezing the protein in a very thin "film" of buffer very quickly to create vitreous ice. Although current approaches work well for some proteins, for many proteins it creates problems that can result in the protein being damaged or fully unfolded. Moreover, the protein can adopt a preferred orientation such that only one view is seen. This can result in artefacts in the structure or often an inability to solve the structure. Many of these problems result from interactions with the interface between the buffer and air (air water interface (AWI)). Our groups have already started to understand some of the affects that can happen, often in a time dependant manner but this is just the tip of the iceberg. This grant will allow us to better understand what happens during sample preparation by looking at how proteins behave (1) over time from sub ms to seconds, (2) on different grids and environments used to support the sample and (3) with different additives added that can change the behaviour of the sample such as detergents to lower surface tension. For our first objective we have a unique piece of equipment that will allow us to make grids from sub ms (not possible to our knowledge by any other group) to seconds. This unique capability coupled with our access to world class EM facilities means we can for the first time look at how the protein behaves over time during the sample preparation process and see how we might minimise some of the negative effects. For the second objective we will see how the chemistry of the support film of the grid which holds the sample and the environmental gas used changes the protein behaviour. We already have pilot data to show a big difference between gold and copper grids indicating that the chemistry of the grid may influence the protein. The third objective will look at how different additives such as detergents influence protein behaviour, we have seen for different proteins that they change their behaviour in the presence of additives and these can make the difference between success and failure but how they work is poorly understood. By better understanding this we may be able to recommend specific additives for different problems during sample preparation. Together this will allow us to better define the rules that affect sample preparation making these experiments more successful to improve data collection and make certain proteins tractable to structure determination.

Cryo-EM has seen enormous growth in recent years but still has further potential if technology and new approaches can be developed around sample preparation, which still creates a bottleneck and can be a fundamental limitation for many proteins. The overarching aim of this work is to provide a detailed understanding of the factors that influence protein behavior and stability in the thin film environment formed during cryo-EM sample preparation and devise evidence-based solutions and robust protocols, applicable over a range of sample types. This timely proposal will take advantage of the increase in speed of data collections and multi-grid EPU with unique technology available in the Muench lab which can produce grids in the sub ms timescale. This work is split into 4 specific objectives. The first will look at how grid preparation across us-s timescales can influence preferred orientation, degradation, concentration, oxidation and stability. Building on existing work, this will provide important information on the ability to mitigate important aspects of sample preparation through decreasing the time of grid preparation and how they might be mitigated in conventional approaches. The second will allow us to understand how different environments influence sample preparation such as the grid support (copper vs gold) and environmental conditions such as atmospheric gas influence sample and droplet behavior. The third will look at how additives such as DTT and detergents influence sample behavior such as preferred orientation and stability, and on sample partitioning in the ice. The final objective will develop a new interface for the cryo-EM community to troubleshoot problems in sample preparation. This work builds on our strong pilot data and robust protocols with ready access to a range of different protein samples. By combining our data from objectives 1-3 we aim to produce a "toolkit" to better inform workflows for researchers from academic and industrial settings.