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.

本研究的冷冻电子显微镜（cryo-electron microscopy，cryo-EM）实验依托阿拉巴马大学伯明翰分校（University of Alabama at Birmingham, UAB）校级研究核心项目开展，并得到Terje Dokland博士与James Kizziah博士的技术支持。赛默飞世尔科技（Thermo Fisher Scientific, TFS）Glacios 2型冷冻电镜搭载Falcon 4i直接电子探测器，通过TFS EPU及断层扫描软件实现高通量冷冻电镜操作与易用性优化。该设备经脱铁铁蛋白单颗粒分析验证，可实现最高约500张/小时的数据采集速度，并具备2.2 Å的分辨率性能。TFS Talos F200C型电镜搭载Ceta-S CMOS相机与Direct Electron Apollo直接电子探测器，主要用于负染样品成像及特种冷冻电镜实验。样品制备环节使用了Pelco EasiGlow辉光放电仪、PIE Scientific TergeoEM等离子清洗仪及FEI Vitrobot Mark IV样品冷冻制样机器人。Gatan 626与698型"Elsa"冷冻样品杆用于Talos F200C的冷冻电镜实验。本研究配备自研GPU加速计算工作站，可借助CryoSPARC Live软件实时处理单颗粒冷冻电镜数据；CEMF通过10Gbps光纤直连UAB超级计算机，用于数据卸载与向用户分发实验数据。细胞于过夜培养至次日清晨后进行传代稀释，继续培养3小时至OD600值达到0.1；随后以8000×g离心2分钟，重悬于磷酸盐缓冲液（PBS）中用于冷冻电镜样品制备。