PT - JOURNAL ARTICLE AU - Benjamin Basanta AU - Marscha M. Hirschi AU - Danielle A. Grotjahn AU - Gabriel C. Lander TI - A case for glycerol as an acceptable additive for single particle cryoEM samples AID - 10.1101/2021.09.10.459874 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.09.10.459874 4099 - http://biorxiv.org/content/early/2021/09/11/2021.09.10.459874.short 4100 - http://biorxiv.org/content/early/2021/09/11/2021.09.10.459874.full AB - Buffer composition and sample preparation guidelines for cryo-electron microscopy are geared toward maximizing imaging contrast and reducing electron beam-induced motion. These pursuits often involve the minimization or complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, we show that inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by a ∼2.3 Å reconstruction of mouse apoferritin (∼500 kDa) and a ∼3.3 Å reconstruction of rabbit muscle aldolase (∼160 kDa) in presence of 20% v/v glycerol. While we found that generating thin ice that is amenable for high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion nor an inability to pick particles. Overall, our findings indicate glycerol should not be discounted as a cryoEM sample buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.Competing Interest StatementThe authors have declared no competing interest.