NIEHS Cryo-EM Core Facility

单粒子冷冻电子显微镜(Cryo-EM)是一种高分辨率确定大分子络合物结构的强有力的技术。它避免了X射线结晶学样品结晶的需要，需要有限数量(纳克)的纯化材料，并可以提供关于大型动态结构的近原子分辨率信息。在过去的三年里，NIEHS一直在与杜克大学和北卡罗来纳大学教堂山分校合作，在低温EM方面建立区域能力。分子显微镜联盟(MMC)被设想为三个成像设施之间的联盟，每个机构一个(杜克大学、北卡罗来纳大学和NIEHS)。该业务由一个紧密联系的核心团队统一起来，提供数据获取、培训和标本准备和图像处理方面的支持。这些设施预计将在两年内投入使用。 NIEHS以创纪录的时间建造了Cryo-EM核心，引领了这一努力。其FEI Talos Arctica低温电子显微镜于2017年6月全面运行，也就是下订单9个月后。在其运作的两年中，来自20多个小组的结构生物学家接受了样品制备方面的培训。本财政年度的样本在200多次筛选会议期间进行了检查，从而确定了适合对几个建筑群进行高分辨率数据收集的条件。最初的结构是从Talos Arctica收集的数据中获得的。 杜克冷冻-EM设施配备了一流的FEI Titan Krios，于2018年4月中旬部署。这大大加快了对NIEHS设施以前优化过的标本的数据收集。在不到四个月的时间里，来自Krios的数据导致了3到5个范围内的另外10个结构的解决方案(表)。自部署以来，该仪器一直以一周七天、每天24小时不间断的数据收集模式运行。许多其他项目正在稳步进行优化过程，预计在不久的将来会产生新的结构。使用他们自己的数据，来自不同群体的合作者接受了利用软件来解决他们自己的结构的培训。在相对较短的一年时间里，几个实验室现在能够独立优化样品制备，一旦收集到高分辨率数据，就可以解决自己的结构问题。这进一步证明了由提供高质量数据的组织支持的协作培训的可行性。 北卡罗来纳大学的设施于2019年春季开业。配备了Talos Arctica低温透射电子显微镜，新增加的设备极大地提高了筛选能力。这使得NIEHS和北卡罗来纳大学的设施都能投入一些时间来优化其200keV仪器的数据收集。这些通常产生3.2A分辨率的结构。 总体而言，低温电磁核心的运行帮助在研究三角园区建立了一个充满活力的低温电磁社区。

Single-particle cryo-electron microscopy (cryo-EM) is a powerful technique to determine the structure of macromolecular complexes at high resolution. It circumvents the need to crystalize samples for X-ray crystallography, requires limited quantities (nanogram) of purified material and can provide near atomic resolution information on large and dynamic structures. Over the past three years NIEHS has been collaborating with Duke and UNC Chapel Hill to establish regional capabilities in Cryo-EM . Molecular Microscopy Consortium (MMC) was envisioned as an association between three imaging facilities, one for each institution (Duke, UNC and NIEHS). The operation is unified by a single closely connected core team providing expertise in data acquisition, training and support in specimen preparation and in image processing. The facilities were expected to become operational in the course of two years. NIEHS led the effort by building the Cryo-EM Core in record time. Its FEI Talos Arctica cryo-electron microscope was fully operational in June 2017, nine months after placing the order. During its two years of operation, structural biologists from over two dozen groups were trained in specimen preparation. This fiscal year specimens were examined during over two hundred screening sessions which lead to the identification of conditions suitable for high resolution data collection for several complexes. Initial structures were obtained from data collected at the Talos Arctica. The Duke cryo-EM facility, equipped with a top-of-the-line FEI Titan Krios, was deployed in mid-April of 2018. This greatly accelerated the data collection on specimens that had been previously optimized at the NIEHS facility. In less than four months, data from the Krios lead to the solution of ten additional structures in the range of 3 to 5 (Table). The instrument has been operating in data collection mode uninterrupted 24 hours a day, seven days a week since its deployment. Many other projects are steadily progressing through the optimization process and are expected to yield new structures in the near future. Using their own data, collaborators from different groups were trained in the utilization of software to solve their own structures. In a relatively short period of one year, several laboratories are now capable of independently optimizing specimen preparation and, once high-resolution data is collected, of solving their own structures. This further demonstrates the viability of collaborative training supported by an organization providing high quality data. The UNC facility opened its doors in the Spring of 2019. Equipped with a Talos Arctica cryo-TEM, the new addition dramatically increases the screening capacity. This has allowed both the NIEHS and UNC facilities to dedicate some time to the optimization of data collection in their 200 KeV instruments. These routinely yield structures at 3.2 A resolution. Overall, the operation of the Cryo-EM core has helped establish a vibrant cryo-EM community at the Research Triangle Park.