SUMMARY Single particle cryoEM is now a ubiquitously employed methodology for high-resolution structure determination of biomedically important macromolecular complexes. CryoEM structures have revealed the detailed mechanisms underlying a wide range of cellular functions and helped understand how environmental or genetic factors perturb biological function to give rise to disease. However, all biological specimens prepared for single particle cryoEM imaging are exposed to the hydrophobic air-water interface during the sample preparation process. This interaction with the air-water interface has a destructive effect on the structural integrity of the targeted specimen, resulting in partial or complete unfolding of proteins, compositional dissociation of complexes, and widespread aggregation of the sample. This interaction with the hydrophobic air-water interface poses the single largest challenge facing the field of single particle cryoEM, and severely limits the utility of cryoEM structure determination for a wide range of samples. Particularly stable samples that maintain structural integrity after repeated interactions with the air-water interface often adopt preferred orientations relative to the hydrophobic surface, which either diminishes or completely eliminates one’s ability to determine a high-resolution structure of the targeted specimen. While there are additives and newer sample preparation devices that aim to mitigate these issues arising from the air-water interface, none of these are broadly applicable for all samples, nor do they fully overcome the air-water interface issues. Thus, cryoEM researchers often spend as much time (or more) optimizing cryoEM sample preparation methods as in establishing expression and purification conditions for a given macromolecule. Given that all commonly used cryoEM sample preparation technologies expose particles to the air-water interface, most of the acquired particle data are damaged and must be computationally discarded during image analysis. This makes cryoEM structure determination incredibly inefficient. We propose to overhaul the grid preparation process to abolish air-water interface interactions during cryoEM grid prep to accommodate preservation of fragile, hard-to-purify samples while substantially improving the efficiency of cryoEM data collection. This new technology will minimize damage to protein samples by blocking interaction with the air-water interface, which will increase applicability and efficacy of cryo-EM data collection, while also enabling robust quantification of macromolecular abundance or conformational dynamics present in a sample. Using a combination of graphene and nanocage technologies we will develop a cheap and robust methodology for cryoEM sample preparation that abolishes air-water interface interactions. Successful execution of the proposed sample preparation platform will advance our understanding of macromolecular mechanisms by enabling the preservation of challenging samples that thus far been recalcitrant to single particle cryoEM studies. This innovative cryoEM grid system will enable researchers to probe unexplored aspects of cell biology, and have a transformative impact that will reverberate throughout the structural biology community.

NARRATIVE Our ability to develop effective therapeutics to treat human diseases is greatly aided by structural descriptions of macromolecules. However, preserving these isolated targets for structural study with one of the most popular methods for structure determination, cryo-electron microscopy, is a major bottleneck in our ability to understand the mechanisms underlying disease. This proposal aims to overhaul our approach to cryo-electron microscopy structure determination in order to overcome this bottleneck, ushering in a new era of structural biology.