Cryo-EM grid preparation using gas dynamic virtual nozzles

Project Summary Recent advances in cryo-electron microscopy (cryoEM), such as the development of direct detectors, automation and 3D particle reconstruction algorithms, have transformed structural biology by enabling investigators to obtain near atomic resolution structures without the need to grow crystals. A significant challenge that limits the wider applicability of cryoEM as a structural tool is the preparation of suitable samples in thin (andlt; 100 nm) layers of vitreous ice in the approximately micron-sized holes of a substrate grid. This arises from the large surface-area-to-volume ratio of the thin aqueous layers prior to freezing of the thin liquid layer in a cryogen. Biological macromolecules preferentially localize to the air-water interface in the thin liquid layer, giving rise to preferred orientations or, in some cases, denaturation. A significant development in overcoming this problem has been the development of droplet based approaches to depositing samples on the grids. This minimizes the dwell time of the protein in the water layer, i.e., the time between spotting of the sample on the grid and plunge- freezing of the liquid layer. The spot-to-plunge time of state-of-the-art instrumentation (~10 ms), however, is still nearly three orders of magnitude longer than the time for diffusion of sample to the air-water interface (~ 10-100 microseconds). The proposed instrument will use thin liquid jets formed using gas dynamic virtual nozzles (GDVN) together with an ultrarapid (~50 m/s) plunging system to reduce the spot-to-plunge time to the microsecond regime. By minimizing this time to less than the diffusion time to the air-water interface, preferential orientation and degradation issues can be minimized. Additionally, the device has the potential to precisely control the ice layer thickness and readily adapt to time-resolved studies. Successful development has the potential to significantly widen the biological macromolecules and scientific questions that can be addressed by high-resolution structure determination using single particle cryoEM.Project Narrative The ability of cryo-electron microscopy to reveal the three-dimensional shape of biological macromolecules to near atomic resolution without the need for crystallization has transformed structural biology. Sample preparation, however, remains a pressing challenge. This proposal aims to develop a microfluidics based approach to ultrafast deposition and freezing of samples to overcome present limitations in sample preparation.