MicroED enables atomic structure determination from nano- or microcrystals, allowing for rapid structural analysis of crystalline powders or mixtures. However, the TEM vacuum limits interrogation of solvated molecular crystals, such as those of proteins or small-molecule hydrates, to frozen-hydrated conditions. This necessitates often lengthy optimization of vitrification conditions, presents challenges for non-aqueous solvents, and does not offer room-temperature insights. Here, we present a strategy for room-temperature MicroED of crystals suspended in solvent, using liquid cells (LCs) comprised of two TEM grids “sandwiched” together with a liquid layer in between. As room-temperature crystals of hydrated organic or biological molecules are sensitive to electron beam damage, ultra-low-dose MicroED data collection methods leveraging a combination of fast event-based electron counting and quasi-serial diffraction acquisition are applied. We examine use-cases of this method by determining a series of room-temperature solvated structures, including those of an antibiotic compound in a pharmaceutically relevant, highly hydrated state, and a hydrated polypeptide. Our results suggest the utility of LC MicroED in visualizing molecular conformations exclusive to a room-temperature solvated state, which would otherwise be inaccessible by conventional MicroED.
Vlahakis et al. (Sun,) studied this question.