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.
Original language | English |
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Pages (from-to) | 38-42 |
Number of pages | 5 |
Journal | Journal of Structural Biology |
Volume | 198 |
Issue number | 1 |
DOIs | |
Publication status | Published - 1 Apr 2017 |
Externally published | Yes |
Funding
Research reported in this publication was supported by the National Institute of General Medical Sciences (NIGMS) under award number 1R01GM120553-01 (to D.V.), 1R01GM118396-01 (to J.M.K.), R01AI070771 to VSR, T32GM008268 (to A.J.B. and A.C.W.). J.S. acknowledges support from the Netherlands Organization for Scientific Research (NWO, Rubicon 019.2015.2.310.006) and the European Molecular Biology Organisation (EMBO, ALTF 933-2015). We are grateful to Neil King (University of Washington) for providing the O3-33 construct.
Keywords
- Cryo-electron microscopy
- Single particle
- Vitrification