Automated cryo-plunger for preparation of vitrified samples for cryo-electron microscopy and cryo-electron tomography

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The atomic resolution of cellular processes has given researchers the ability to understand the molecular details of how biological processes work. Traditionally, crystallography has been the predominant technology used to determine structures; however, one of the bottlenecks of this technique is the requirement to produce 3D protein crystals. Recently, single-particle cryo-electron microscopy (cryo-EM) has undergone a `resolution revolution' and has become one of the leading methods solving the structures of macromolecular complexes. Cryo-EM is now routinely able to determine structures in the resolution range of 1.5-3.0 Å, without the need for crystals. The process of developing a new single-particle cryo-EM project begins with the preparation and characterization of the protein sample. The protein must be transferred onto an EM grid, blotted with a filter paper to produce a thin aqueous layer, and then instantaneously vitrified in liquid ethane. Achieving perfect vitrification is difficult and challenging to reproduce if performed manually. Here we request funds to support the purchase of a state-of-the-art Thermo Scientific Vitrobot Mark V automated cryo-plunge instrument. The Vitrobot Mark V has been re-designed; it now represents a technical advancement in EM grid preparation that allows researchers to plunge-freeze pre-clipped EM grids, as well as to perform single-sided blotting. The new design allows faster vitrification compared to the older Vitrobot models and is designed to yield consistent, high-quality reproducible results. Currently, this instrument would be the first of its kind in Canada. The Vitrobot Mark V will benefit the entire structural biology community in Toronto and will expand the current capabilities of the University of Toronto Microscopy Imaging Laboratory.