A next-generation energy filter for electron cryotomography at Imperial College

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The function of the molecular machines of life is intimately associated with their context inside cells. Many multiprotein complexes are dynamic, fragile, or membrane-associated, making them challenging or impossible to effectively study using the traditional structural biology approaches that involve protein purification and characterization by X-ray crystallography, nuclear magnetic resonance, or single particle analysis cryoEM. Studying molecular machines in situ is therefore crucial for us to fully understand their mechanisms and will form a foundation for the development of the biological sciences into the 21st century. Such foundational work will also have application in visualizing the mechanisms of diseases and pathogens in situ, the cellular ramifications of the action of pharmaceutical interventions, and the synthetic design (or co-option of existing) molecular machines. The only way to visualize the structural molecular biology of molecular machines in situ is by using electron cryotomography, which co-opts the existing power of electron cryomicroscopes (e.g., with the 2017 Nobel Prize in Chemistry) to image unique biological samples such as cells from a range of angles to reconstruct their 3-D structures and to resolve their molecular components: effectively a "nanoscale CT scan". These 3-D images, or tomograms, disentangle the complexity of the cell that would otherwise be overwhelming in a 2-D image; similar structures (e.g., protein complexes) can subsequently be aligned, classified, and averaged, to arrive at 3-D structural snapshots of their action in situ.Our proposal is to purchase a next generation "energy filter" to enable routine electron cryotomography at Imperial College London. This purchase will propel Imperial from being "behind the times" to "internationally competitive" with a single upgrade purchase and catalyse development of diverse projects already nascent in our various CoI's work.