The field of cryogenic electron microscopy (cryo-EM) has rapidly been gaining traction as astructural characterization technique for proteins. Sample preparation for cryo-EM isadvantageous over other proven techniques like x-ray crystallography as proteins do not need tobe crystallized for high-resolution structural analysis. While the cryo-EM sample preparationtechnique keeps proteins in their native form, cryo-EM suffers from a bottleneck in both qualityand throughput of samples. Recent advances in the field have focused on increasing samplethroughput through automated preparation techniques. However, the more important problem incryo-EM samples now has become protein damage from the air-water interface. Before vitreousfreezing, proteins in the sample droplet collide and stick to the droplet surface, where theydenature to form an unraveled monolayer. The more time the sample is exposed before freezing,the more time proteins have time to interact with the surface and denature. Current commercialsystems fail to directly address the problems associated with the interface, focusing instead onthe automated production of samples with thin, vitreous ice. While these improvements arevaluable to the overall goals of reducing ice thickness, they ignore damage to the samplemolecules themselves, preventing users from repeatably obtaining high-resolution structures.The proposed project aims to revolutionize sample preparation techniques for cryo-EM byexploring a method of generating higher quality samples that exhibit significantly less damagefrom the air-water interface. The proposed system aims to limit sample exposure to the air-waterinterface by reducing the rate of freezing by an order of magnitude. In order to reduce damagefrom the air-water interface with high quality samples, the system will aim to vitrify samples in lessthan 10 ms with a large percentage of ice suitable for high-resolution imaging. This "fast freezing"system also is also conceived to populate sample grids with consistently thin ice by reducing theamount of excess water that is frozen on the grid, creating larger areas of uniform ice andbiomolecular sample per grid. Hummingbird Scientific aims to develop and test the theory behindthe system in Phase I.
Public Health Relevance Statement: Project Narrative Project Title: Development of an Instant Freezing Sample Preparation System for Cryo- EM Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Norman Salmon Narrative: The proposed fast-freezing system technology has the potential to improve the efficiency and accuracy of cryogenic electron microscopy (cryo-EM) by reinventing the sample preparation process. By removing common sources of sample inconsistency, this new technique will allow users to more easily and accurately contribute high resolution biomolecular structures to protein structure data banks to be utilized by researchers in fields such as drug analysis and discovery, virology, and cancer research. Acceleration of health-related solutions that require knowledge of biomolecular structure and function is the primary goal of this work.
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