Journal
FRONTIERS IN MOLECULAR BIOSCIENCES
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmolb.2022.903148
Keywords
cryoEM specimen preparation; vitrification; automation; self-wicking grids; air-water interface issues; preferred orientation; denaturation; dissociation
Categories
Funding
- NIH [GM126982, 2017246757]
- NSF GRFP [5T32GM007287]
- Canadian Institute for Advanced Research Bio-Inspired Solar Energy program
- HHMI Transformative Technology 2019 Award
- EPSRC
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The specimen preparation process is crucial for the success of cryo electron microscopy (cryoEM) structural studies. Traditional manual methods have limitations in speed and efficiency, while new automated technologies offer improved controllability and stability. As early users of the chameleon system, we share our experiences and lessons learned through case studies, providing recommendations for future users and the field of cryoEM specimen preparation.
The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The process has transformed structural biology, but it is largely manual and can require extensive optimisation for each protein sample. The chameleon instrument with its self-wicking grids and fast-plunge freezing represents a shift towards a robust, automated, and highly controllable future for specimen preparation. However, these new technologies require new workflows and an understanding of their limitations and strengths. As early adopters of the chameleon technology, we report on our experiences and lessons learned through case studies. We use these to make recommendations for the benefit of future users of the chameleon system and the field of cryoEM specimen preparation generally.
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