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*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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 languageEnglish
Pages (from-to)38-42
Number of pages5
JournalJournal of Structural Biology
Volume198
Issue number1
DOIs
Publication statusPublished - 1 Apr 2017
Externally publishedYes

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

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