Development of Tools to Improve Sample Preparation and Outcomes in Cryogenic Electron Microscopy Single Particle Analysis and Micro-electron Diffraction

Atomic structures of biomolecules and biomolecular complexes facilitate detailed understanding of biomolecular function. They are key drivers of the ongoing revolution in molecular biology and of many areas of biotechnology, including development of pharmaceuticals, of enzymes and light harvesters for bioenergy, and of engineered organisms for bioremediation. As a result of recent advances in the technical capabilities of electron microscopes and detectors, single- particle cryo-electron microscopy (cryo-EM) has emerged as a powerful approach to obtaining near-atomic- resolution structures of large biomolecular complexes, membrane proteins, and other targets of major scientific interest to bioenergy, including targets that have been intractable to other structural methods. The primary obstacle to obtaining high resolution structures are now most often associated with sample preparation. This Phase I SBIR project will focus on two key sample preparation challenges in single-particle cryo-EM: obtaining sample films of a target thickness on cryo-EM support foils to maximize image contrast; and reproducibly cooling these samples to obtain largely vitrified ice and minimal beam-induced motion suitable for high resolution imaging. Advances in electron microscopes and detectors have also greatly enhanced the capabilities of micro- electron diffraction (micro-ED), allowing atomic resolution structures to be determined from nanometer size crystals that have been intractable to X-ray-based methods. Key challenges here are again associated with sample preparation and mounting for efficient data collection. To obtain sample films of a target thickness for single-particle cryo-EM, methods will be developed to fabricate nanometer-thick sample support foils having contact line pinning steps and holes of different depth, and the resulting dynamics and thicknesses of dispensed liquids will be compared with those on conventional foils. To uniformly deposit very small amounts of sample liquid over the active area of the foil and to remove excess liquid, deposition devices based on micropillar arrays will be developed. To improve sample cryocooling outcomes, the detailed processes that occur as a cryo-EM grid enters a liquid cryogen will be studied, with emphasis on interfacial interactions between liquid cryogen and sample support materials. Drawing on MiTeGen’s broad expertise in developing sample supports for X-ray crystallography, novel concepts for foils and grids to address sample handling in micro-ED will be evaluated. The proposed work, if successful, will lead to commercial development of sample supports and sample deposition systems with potential to substantially increase the throughput of cryo-EM structure determination pipelines, allow much more efficient use of expensive cryo-TEM facilities, and improve overall data quality. This will support cryo-EM studies of key biomolecular structures and interactions used by microbes and plants relevant to bioenergy and bioremediation, topics of major interest to the DOE. This project and the manufacture and sale of the products it generates will provide good paying jobs and support economic development in the Southern Tier of New York State, which includes many of the state’s most economically depressed counties and cities.