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

This method involves performing several successive rounds of sample application and blotting before plunge freezing. We tested up to four blotting rounds and observed a marked additive effect of particle density at each step. All sample applications and blotting steps can be carried out on the lab bench, similarly to what is done for negative staining sample preparation, until the final sample application, at which point the tweezers are mounted in a plunge-freezing apparatus to perform the final blotting step using standard procedures. Alternatively, all rounds of sample application and blotting can be performed with the tweezers mounted in a plungefreezing apparatus, if desired. We used Whatman No. 1 filter paper, which is the only extra requirement over our standard grid preparation procedure. For all examples that follow, samples were vitrified using Protochips C-flat grids and imaged on an FEI T12 electron microscope equipped with a Gatan US4000 CCD camera, an FEI TF20 or an FEI Titan Krios, equipped with a Gatan K2 direct detector (Li et al. 2013) . We used MotionCorr2 (Zheng et al. 2016) for movie frame alignment and binned all images by a factor of 4 for visualization.