Development of Thin-Ice Tem Grids to Control the Ice Thickness for Cryo-Electron Microscopy

Abstract

Cryo-electron microscopy (cryo-EM) is a powerful technique to determine structures of biological macromolecules and complexes embedded in a thin layer of vitreous ice. More than 400 protein structures have been determined to be better than 5-A resolutions in the EM databank. In cryo-EM, one limiting factor in image contrast is the ice thickness. Experiments have shown that by extensive efforts to optimize the vitrification process, the contrast of recorded cryo-EM images increased dramatically. Currently, the vitrification process is still a trial and error method, and there is very poor control of the ice thickness. In this study, a new type of TEM grid, termed as Thin-ice TEM grid, will be developed using nanofabrication processes. The thickness of the spacer layer on a Thin-ice TEM grid determines the thickness of the liquid layer, thus the thickness of the vitreous ice. This makes the vitrification process a well-controlled step, and guarantees the ice thickness to be the desired value. In addition, the sample volume is decreased by a factor of 100-10,000. The Thin-ice TEM grid not only eliminates the intensive labor and cryo-EM involvement, but also results in maximized contrast (i.e. signal-to-noise-ratio, SNR) of recorded cryo-EM images and higher resolution of the determined biological structure. This will increase the efficiency and success rate of cryo-EM in structural biology, speed up the understanding of biological functions at molecular level, and potentially facilitate the development of new therapeutic agents to treat diseases in broad areas.