Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids
By Joost Snijder, A. J. Borst, Annie M. Dosey, A. Walls, A. Burrell, V. Reddy, J. Kollman, D. Veesler. 2016
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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.
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Citations in the corpus
- Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus (2019, methodology): “A multiple blotting strategy was employed, as previously described (Snijder et al., 2017).”
- Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors (2018, unknown): “Cryo-EM grids were prepared under > 80% humidity at 4"C inside a cold room, and a multi-blotting approach was used to increase particle density (Snijder et al., 2017).”
- Cryo-EM analysis of a membrane protein embedded in the liposome (2020, methodology): “To increase the proteoliposome density, we implemented a multiapplication method to add protein solution repetitively before final blotting (38).”