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

In conclusion, using a simple and cost-effective trick, one can increase the applicability of single particle cryoEM to protein complexes that cannot be purified in large quantities and/or that behave poorly upon vitrification. Multiple steps of sample application can force particles into the vitreous iced-filled holes of a cryoEM grid to minimize sample requirements for structural studies. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/094623 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/094623 doi: bioRxiv preprint Figure 6 . Multiple rounds of sample application and blotting of HIV envelope glycoprotein ectodomain in the presence of 85 µM dodecyl-maltoside. Scale bars: 100 nm.