Molecular imaging of in situ cellular machinery by cryo focused ion beam milling

Context The molecular machines of living cells are essential for life. Correct function of these machines supports accurate development, physiological function and healthy ageing. Conversely, disruption of pathogen machinery can be used to tackle global health and food security threats, including antibiotic resistance and agricultural diseases. The ISMB is internationally recognised as a centre of excellence for the study of molecular machines, but most of our current work is undertaken on machinery that has been extracted from its cellular location and purified away from other cellular components. While such work provides crucial detailed information about how these macromolecular complexes operate, it misses out on the cell or tissue context in which they work. Imaging these machines in their functional environment is critical and provides unique insight into the context in which they act. However, cells and tissues are many times larger and more complex than the machines themselves. As a result, precise and specific sample preparation is needed to preserve the context of the machinery of interest, to help locate the machinery within the complex cellular environment and to enable the machinery to be imaged using high resolution electron microscopy. The requested cryo focused ion beam (cryo-FIB) microscope will allow thinning of such complex samples, providing a window into cells and tissues to reveal molecular machine organisation and functional context. The research the equipment will enable Specific projects that will benefit from this new equipment include: 1) Structural and mechanistic analysis of transcription-translation coupling; 2) Studies of the molecular machinery that controls the trafficking of cargo and metabolites through cells; 3) Studies of the malaria parasite that will uncover the mechanisms by which the parasite adapts to its different hosts, allowing parasite transmission; 4) Studies of the molecular mechanisms of bacterial conjugative transfer, the main means by which antibiotic resistance genes spread among bacteria; 5) Studies to elucidate the structural mechanisms underlying co-translational folding and cellular proteostasis; 6) Studies of regulatory proteins whose disruption by naturally occurring mutations cause age-related protein polymerisation that perturbs liver and lung cell function; 7) Studies of the contribution of the actin cytoskeleton to membrane remodelling during muscle development; 8) Studies of amyloid fibre assembly and disassembly that will reveal how the cell's quality control machinery handles protein misfolding associated with diseases of ageing. Aims and objectives Our overall objectives are 1) for the EM Facility team to establish efficient and robust workflows using the new cryo-FIB microscope; 2) to train both established and non-expert users to apply cryo-FIB workflows to current and new projects in BBSRC-relevant areas; 3) for users to share best-practice with the user community via CCPEM and other training networks. The new cryo-FIB microscope, together with our current equipment will provide researchers with a full workflow which spans biological scales, from tissues to atoms. This will be game-changing for EM Facility users and will be one of only a few such research facilities in the UK. Potential applications and benefits The research that will be conducted using the cryo-FIB microscope aligns with the UKRI-BBSRC Delivery plan by aiming at understanding the rules of life and providing an integrated understanding of health. This research will also exploit new ways of working, including the increasing implementation of machine learning methods to enhance the in situ molecular structure determination pipeline.