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TitleVitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids.
Journal, issue, pagesJ Struct Biol, Vol. 198, Issue 1, Page 38-42, Year 2017
Publish dateFeb 22, 2017
AuthorsJoost Snijder / Andrew J Borst / Annie Dosey / Alexandra C Walls / Anika Burrell / Vijay S Reddy / Justin M Kollman / David Veesler /
PubMed AbstractSingle 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.
External linksJ Struct Biol / PubMed:28254381 / PubMed Central
MethodsEM (single particle)
Resolution7.3 - 7.8 Å
Structure data

EMDB-8613:
3D-reconstruction of O3-33 from a grid prepared with a single round of blotting
Method: EM (single particle) / Resolution: 7.8 Å

EMDB-8614:
3D-reconstruction of O3-33 from a grid prepared with multiple rounds of blotting
Method: EM (single particle) / Resolution: 7.3 Å

Source
  • unidentified (others)

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