Cryo-Electron Microscopy

Cryo-electron microscopy was performed with the help of the UAB institutional Research Core Program with the help of Dr. Terje Dokland and Dr. James Kizziah (University of Alabama at Birmingham). A Thermo Fisher Scientific(TFS) Glacios 2 equipped with a Falcon 4i direct electron detector is optimized for high-throughput cryo-EM and ease of use <i>via </i>the TFS EPU and Tomography software. It has demonstrated data collection speeds up to ~500 images/hour and a capability of 2.2 Å resolution <i>via </i>single particle analysis with apoferritin. A TFS Talos F200C equipped with a Ceta-S CMOS camera and a Direct Electron Apollo direct electron detector is used for imaging of negatively stained samples and specialized cryo-EM applications.For sample preparation, a Pelco EasiGlow glow discharge machine, a PIE ScientificTergeoEM plasma cleaner, and FEI Vitrobot Mark IV sample vitrificationrobot were used. Gatan 626 and 698 “Elsa” cryo-holders are used for cryo-EM on the Talos F200C. An in-house GPU-accelerated computing workstation is used for on-the-flyprocessing of single particle cryo-EM data with CryoSPARC Live, and the CEMF has a direct 10Gbps fiberlink to the UAB supercomputer for offloadingand distribution of data to users. Cells were grown overnight until the morning when they were back diluted and grown for 3 hours until an OD600 of 0.1 was reached, and cells were centrifuged at 8,000 <i>g </i>for 2 minutes and resuspended in PBS for CEM sample prep.