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

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.

Determining the structures of molecular machines in situ is crucial to the biological sciences in the 21st century. Such structural determination are foundational to understanding biological mechanisms, disease processes, drug action, and providing clues to designing our own synthetic molecular machines. Electron cryotomography (cryoET) with subsequent subtomogram averaging is the only general-purpose in situ technique capable of delivering this goal.

We propose to enhance our recently ordered Wellcome Trust-funded electron cryomicroscope (a Thermo Fisher Scientific Glacios microscope with Falcon 4 camera) with a new Thermo Fisher Scientific Selectris energy filter to enable cryoET at Imperial for all users, nucleated by a core of cryoET -curious users with projects awaiting cryoET capability. We believe this equipment will have a lifetime of at least 10 years. The Glacios with Falcon 4, which came on the market recently, promises to be a stellar screening microscope and is likely to enable routine atomic-resolution structure determination of purified biological macromolecules. By adding a cutting-edge Selectris filter (which came on the market in 2020), coupled with the use of cutting-edge software we aim to increase both throughput and resolution by an order of magnitude for subnanometre-resolution structural determination of molecular machines in situ. We anticipate achieving near-atomic resolution for atomic modelling for optimal, abundant samples.

Combined with Lead PI Beeby's high throughput cryoET expertise we project being able to initially acquire many hundreds of tomograms per day, and anticipate increased throughput with new developments on the horizon.

The only equipment capable of cryoET at Imperial is currently our previous-generation FEI Tecnai F20 with Falcon 2 camera, now discontinued and widely considered not viable for cryoET. While we have pushed this instrument to its limit, its resolution is limited to tens of nanometres.