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

We used the mouse hepatitis virus (MHV) spike (S) glycoprotein ectodomain (Walls et al. 2016a; Walls et al 2016c) to illustrate the usefulness of our approach when vitrifying samples in presence of detergents (see Figure 5) . A single round of sample application and blotting of the MHV S ectodomain in the presence of 0.01% NP40 yielded few particles in suspended vitreous ice, even at concentrations up to 4 mg/mL. After the second round of blotting, many more particles could be observed in the ice. We would like to emphasize that in the absence of detergent, a single sample application resulted in a very dense distribution of particles, even at less than half the concentration. We obtained similar results with the HIV envelope glycoprotein ectodomain in the presence of 85 µM of dodecyl-β-D-maltoside (a sample known to be prone to aggregation upon vitrification, Lyumkis et al 2013). Although few particles were detected in vitreous ice after a single round of blotting, good particle density was achieved after the second sample application, thereby alleviating the need for large amount of protein ( Figure 6 ).