Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids

Joost Snijder; Andrew J. Borst; Annie Dosey; Alexandra C. Walls; Anika Burrell; Vijay S. Reddy; Justin M. Kollman; David Veesler

Whereas sample requirements are less restrictive in cryoEM compared to X-ray crystallography or nuclear magnetic resonance, successful vitrification of protein complexes remains a major bottleneck (Grassucci et al. 2007) . A solution of the particles under study is pipetted onto holey grids made of carbon-coated copper or gold (Quispe et al 2007; Russo et al 2014) . The holes in the grid support film become filled with particles suspended in buffer and the excess solution is blotted away using filter paper. The grid is then immediately plunged into a bath of liquid ethane, maintained at liquid nitrogen temperature (-180ËšC) to vitrify the protein solution and preserve it in a near-native frozen-hydrated state (Dubochet 1988). Numerous specimens, however, are reluctant to populate the holes and remain adsorbed on the carbon support film instead, even after significantly increasing the sample concentration. Furthermore, many protein complexes are precious, due to the difficulty to purify them in large quantities, such that using higher concentrations is not an option.