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

细胞过程的原子分辨率使研究人员能够了解生物过程如何工作的分子细节。传统上，晶体学一直是用于确定结构的主要技术；然而，该技术的瓶颈之一是需要产生3D蛋白质晶体。近年来，单粒子低温电子显微镜（cryo-EM）经历了一场“分辨率革命”，已成为解决大分子复合物结构的主要方法之一。Cryo-EM现在通常能够在1.5-3.0 Å的分辨率范围内确定结构，而不需要晶体。开发一种新的单颗粒低温电镜项目的过程始于蛋白质样品的制备和表征。蛋白质必须转移到电磁网格上，用滤纸吸干，形成一层薄薄的水层，然后立即在液态乙烷中玻璃化。实现完美的玻璃化是困难的，具有挑战性的再现，如果进行手动。在此，我们请求资金支持购买最先进的Thermo Scientific Vitrobot Mark V自动低温骤降仪器。Vitrobot Mark V被重新设计；它现在代表了EM网格制备的技术进步，使研究人员能够快速冷冻预剪切EM网格，以及进行单面印迹。与旧的Vitrobot模型相比，新设计允许更快的玻璃化，并旨在产生一致的，高质量的可重复结果。目前，这种仪器将是加拿大同类仪器中的第一种。Vitrobot Mark V将使多伦多的整个结构生物学社区受益，并将扩大多伦多大学显微镜成像实验室的现有能力。

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.