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

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.

Resolving the dynamic changes within macromolecules at atomic resolution is key to understanding mechanism but this information is lost in the "static" snapshots obtained by conventional structural biology techniques. Although the x-ray free electron laser (XFEL) has provided unprecedented detail of reaction mechanisms in the ps timeframe and Å resolution, with NMR, RAS and EPR spectroscopy capable of similar temporal resolutions, for many systems, including large protein complexes and membrane proteins these approaches are not sufficient. Time-resolved electron microscopy (EM) is ideally suited to these systems and spans a huge breadth of biological sciences but is currently underexploited with only a handful of machines world-wide producing results, typically in the >50 ms time-frame. These studies are traditionally hindered by poor reproducibility, grid quality, data collection speeds, resolution limits and the ability to computationally sort different conformational states. Importantly, with recent developments in the EM field these challenges can now be met.
This proposal will address inconsistencies in grid quality (objective 1) and use a new cutting edge microfluidic device (objective 2) to generate pilot data on the V-ATPase mechanism (objective 3), generating a prototype setup which can work in the >5 ms resolution. Grid quality will be addressed by using a new micro-fibre grid, specifically designed to work with micro-droplet dispersion and testing different support films. The current rapid mixing device is unreliable, prone to blockages and difficult to assemble. Therefore a new collaboration with the Trebbin group in Hamburg will adapt the cutting edge microfluidic mixer devise currently designed for the XFEL and time-resolved SAXS to time-resolved EM, providing a robust and fast time-resolution device. The resulting prototype will generate pilot data on the vacuolar ATPase providing proof of principal which will underpin further funding.

The design and objectives of the proposed project are focused on both the development of time-resolved electron microscopy (EM) and its availability to the wider community. Ongoing conversations with experts in the EM field and academia have informed on the need for this step-change in our ability to understand protein function. The data generated over the course of this project can be used by a wide spectrum of academia and industry, for example in informing on the plasticity of inhibitor binding sites for structure based inhibitor design. Impact will be generated through the following routes;

With the rapid expansion in EM has come a shortage of researchers with the skills required for all aspects, particularly cryo-EM data collection and analysis. This project will equip a postdoctoral researcher at the cutting edge of time-resolved EM studies in addition to the skills to carry out standard cryo-EM studies, thus increasing the long-term impact of the project. The nature of this work makes it an ideal starting point for a junior fellowship application and Dr Muench (SM) has already identified very talented and enthusiastic PDRA's who would welcome the opportunity to develop a novel angle in EM studies for this purpose.

We have strong links with industry; SM has students with Pfizer and Meddimune and has given an invited talk at GlaxoSmithKline on his EM work and possible collaborations. Moreover, SM has been invited to speak at the upcoming SCI conference which will be attended by a wide range of industrial partners. As part of the Astbury centre SM is the lead for the ion channel research industrial hub which directly interfaces with industry on a regular basis. The significant investment by Leeds in two Titan Krios microscopes has given us strong links to FEI, which make the current Vitrobot Mark IV freezing apparatus. We will continue to maintain our links to industry to ensure that both the direct results and the wider methodologies will achieve their full impact within this sector.

In addition to the academic and scientific community SM has a strong track record in public engagement and the nature of this work makes it translatable to this forum. Through events like the Astbury conversation, discovery zone and college visits, this work will be used to form the basis of "inspiring the next generation of scientists" and informing the public on the cutting edge science being carried out through BBRSC research funding.

Upon dissemination of successful research outputs, we strongly anticipate an expansion in our collaborative research in this field with both industrial and academic partners worldwide. We have an extensive collaborative network, many of whom have already shown an interest in the technology and have systems that would greatly benefit from time-resolved studies (many are BBSRC funded). We will reach out to others in the direct field of time-resolved studies through the attendance of relevant conferences, for example the recent COST conference on time-resolved methodologies (through which Dr Trebbin and SM met), NRAMM and Gordon research conferences. Through this network we hope to drive the establishment of a world-class structural biology tool and therefore deliver an intellectual and economic impact to U.K. and global research.

In summary this work translates strongly into the structural biology, enzymology, pharmacology, medicinal chemistry, and protein dynamics fields. Through regular conference attendance, our extensive network (both academia and industry), rapid publication (were appropriate) and use of social media we will maximise this impact. The visual nature of this work, the broad scope and its association with improving health will make strong material for engaging the public and increasing their understanding and confidence in U.K. scientific research.