NIEHS Cryo-EM Core Facility

NIEHS 冷冻电镜核心设施

• 批准号: 10008872
• 负责人: Mario Borgnia
• 金额: $ 163.65万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10008872
• 关键词: Communities; Complex; Computer software; Core Facility; Cryoelectron Microscopy; Crystallization; Data; Data Collection; Data Quality; Electron Microscope; Electron Microscopy; Ensure; Future; Goals; Hour; Institution; Laboratories; Lead; Macromolecular Complexes; Membrane Proteins; Methods; Microscopy; Mission; Molecular; Molecular Structure; National Institute of Environmental Health Sciences; Nuclear Magnetic Resonance; Preparation; Process; Publications; Research; Resolution; Sampling; Specimen; Structural Biologist; Structure; Techniques; Time; Titan; Training; Training Support; X-Ray Crystallography; collaborative environment; data acquisition; image processing; imaging facilities; instrument; macromolecule; new technology; novel strategies; operation; particle; process optimization; response; screening; structural biology; tool

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.

单粒子低温电子显微镜（cryo-EM）是一种高分辨率测定大分子复合物结构的强大技术。它避免了需要结晶样品的X射线晶体学，需要有限数量（纳克）的纯化材料，并可以提供近原子分辨率的大型和动态结构的信息。在过去的三年里，NIEHS一直与杜克和查佩尔山合作，建立低温EM的区域能力。分子显微镜联盟（MMC）被设想为三个成像设施之间的协会，每个机构（杜克，杜克大学和NIEHS）一个。该操作由一个紧密联系的核心团队统一进行，该团队提供数据采集、培训和样本制备和图像处理方面的支持方面的专业知识。这些设施预计将在两年内投入使用。 NIEHS在创纪录的时间内建立了Cryo-EM Core。其FEI Talos Arctica冷冻电子显微镜于2017年6月全面投入使用，即下订单9个月后。在两年的运作中，来自二十多个团体的结构生物学家接受了标本制备方面的培训。本财政年度，在200多个筛选会议期间对标本进行了检查，从而确定了适合于几个复合体的高分辨率数据收集的条件。最初的结构是从在北极塔罗斯收集的数据中获得的。 配备顶级FEI Titan Krios的杜克冷冻EM设施于2018年4月中旬部署。这大大加快了先前在NIEHS设施优化的样本的数据收集。在不到四个月的时间里，来自Krios的数据导致了3到5范围内的十个额外结构的解决方案（表）。该仪器自部署以来一直以数据收集模式运行，每周7天、每天24小时不间断。许多其他项目正在通过优化过程稳步推进，预计将在不久的将来产生新的结构。利用他们自己的数据，来自不同群体的合作者接受了利用软件解决他们自己结构的培训。在相对较短的一年时间内，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，就能够解决自己的结构。这进一步表明，在提供高质量数据的组织的支持下开展合作培训是可行的。 该工厂于2019年春季开业。配备了Talos Arctica cryo-TEM，新增加的设备大大提高了筛选能力。这使得NIEHS和NIEHS设施都能够投入一些时间来优化其200 KeV仪器中的数据收集。这些通常产生3.2 A分辨率的结构。 总的来说，Cryo-EM核心的运作有助于在三角研究园区建立一个充满活力的cryo-EM社区。