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

To demonstrate the efficacy of this method, we used a computationally designed octahedral protein cage (King et al. 2012) , assembled from 24 identical subunits, as a test specimen (O3-33, Figure 1 ). At 1 mg/mL, the O3-33 solution yielded an optimal particle density for single particle cryoEM imaging. Diluting the sample five-or tenfold significantly reduced the distribution of particles over suspended ice. This effect could be partially overcome for both dilutions after a second round of sample application and blotting. After the fourth blotting step, the particle density in suspended vitreous ice became comparable to that observed with grids prepared with a single application of a ten times higher protein concentration. These results illustrate that the method can greatly alleviate sample requirements for single particle cryoEM, both in terms of protein concentration and total amount required.