title: Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids.
abstract: Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Joost
authors.initials: J
authors.lastname: Snijder
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Borst
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Annie
authors.initials: A
authors.lastname: Dosey
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Walls
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Anika
authors.initials: A
authors.lastname: Burrell
authors.affiliation: Department of Integrative Computational and Structural Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Vijay S
authors.initials: VS
authors.lastname: Reddy
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Kollman
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA. Electronic address: dveesler@uw.edu.
authors.forename: David
authors.initials: D
authors.lastname: Veesler
mesh_terms: D020285:Cryoelectron Microscopy / Q000191:economics / Q000379:methods*; D005615:Freezing; D046911:Macromolecular Substances; D008722:Methods; D013048:Specimen Handling; D058989:Vitrification
chemical_list: D046911:Macromolecular Substances
keywords: Cryo-electron microscopy; Single particle; Vitrification