National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM120553-01
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
R01AI070771
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM118396-01
米国
Netherlands Organization for Scientific Research
NWO, Rubicon 019.2015.2.310.006
オランダ
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
T32GM008268
米国
EMBO
ALTF 933-2015
オランダ
引用
ジャーナル: J Struct Biol / 年: 2017 タイトル: Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids. 著者: Joost Snijder / Andrew J Borst / Annie Dosey / Alexandra C Walls / Anika Burrell / Vijay S Reddy / Justin M Kollman / David Veesler / 要旨: 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 ...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.