CU Boulder Center for Cryo-ET (CCET)

总结： 我们将根据RFAs RM-19-009和-010在更大的网络内建立卫星服务中心， 该中心名为CU-Boulder Center for Cryo-Electron Tomography（CCET）。我们的卫星将聚焦于低温- 用于细胞、细胞器和大细胞的标本制备和冷冻电子断层扫描（冷冻ET） 超分子组装体申请人联合收割机结合了三个私人侦探的专业知识，迈克尔·斯托威尔（高- 分辨率低温电子显微术（cryo-EM）），KarolinLuger（大型复杂组件的生物化学， 尤其是蛋白质-核酸相互作用）和Andreas Hoenger（前P41主任，冷冻EM和冷冻ET 从大分子到细胞和组织）。凭借我们现有的员工，我们将联合收割机广泛的专业知识与冷冻ET 相关软件。我们将提供广泛的冷冻电子显微镜（cryo-EM）和冷冻ET 细胞结构和细胞内大分子组装的准备程序，以及培训 为那些希望在自己的家园建立这些技术或至少部分技术的科学家提供了机会 labs.在博尔德，我们有几个高端显微镜可用，包括新安装的Titan-Krios。 我们的三台显微镜配备了直接电子探测器。此外，我们有一个全面的 用于低温标本制备、存储和处理资源的外围设备，包括计算工具。我们 将增加低温聚焦离子束（cryo-FIB）研磨，以产生细胞和组织的玻璃化薄片。我们提出 具体目标1：提供Cryo-ET样品制备方法 服务：我们的技术和专业知识将提供给我们的用户和进一步的合作 与并行RFA（RM-19-010）中建议的数据收集中心。目标二：交叉训练 服务用户的机会：我们将为用户提供全面的培训，包括一对一的培训 显微镜和相关设备的说明。在适当的情况下，我们将把用户分成几个小组， 一个培训周，作为一个动手操作的设备研讨会，以及由我们的设施开发的相关软件，如 如Serial-EM、IMOD和PEET。虽然培训的重点将放在标本的实践方面， 准备和资料收集时，我们经常遇到新手用户强烈需要一个彻底的介绍， 图像分析软件。具体目标3：设施的管理和组织： 通过全网申请流程分配给我们的人员将有机会参加标本 我们将继续改进过程，并接受有关冷冻ET过程的培训。具有较大潜力的项目将 将高吞吐量数据收集提交给网络的中央集线器设施。科罗拉多的博尔德市 位于丹佛国际机场，交通便利。

Summary: We will establish a satellite service center within a larger network according to RFAs RM-19-009 and -010, named the CU-Boulder Center for Cryo-Electron Tomography (CCET). Our satellite will focus on cryo- specimen preparation and cryo-electron tomography (cryo-ET) applied to cells, organelles and large supramolecular assemblies. The applicants combine the expertise of three P.I.s, Michael Stowell (high- resolution cryo-electron microscopy (cryo-EM)), Karolin Luger (biochemistry of large complex assemblies, especially protein-nucleic acid interactions), and Andreas Hoenger (former P41 director, cryo-EM and cryo-ET from macromolecules to cells and tissues). With our current staff, we combine extensive expertise with cryo-ET and relevant software. We will offer a broad range of cryo-electron microscopy (cryo-EM) and cryo-ET preparation procedures for cellular structures and intracellular macromolecular assemblies, as well as training opportunities for scientists who would like to establish these technologies, or at least part of them, for their home labs. Here in Boulder, we have several high-end microscopes available, including a newly installed Titan-Krios. Three of our microscopes are equipped with direct electron detectors. In addition, we have a comprehensive periphery for cryo-specimen preparation, storage, and handling resources, including computational tools. We will add cryo-Focused Ion Beam (cryo-FIB) milling to produce vitrified lamellae of cells and tissues. We propose the following organization of the facility: Specific Aim 1: Cryo-ET specimen preparation methods offered for service: Our technology and expertise will be made accessible for our users and for further collaborations with the data collection hub proposed in a parallel RFA (RM-19-010). Specific Aim 2: Cross-training opportunities for service users: We will offer comprehensive training for users, including one-on-one instruction on microscopes and related equipment. As appropriate, we will pool users into small groups and run a training week as a hands-on workshop on equipment as well as related software developed by our facility such as Serial-EM, IMOD and PEET. While the emphasis with training will be on practical aspects of specimen preparation and data collection, we often encounter novice users in strong need for a thorough introduction into image analysis software as well. Specific Aim 3: Administration and organization of the facility: Users, assigned to us via the network-wide application process will have the opportunity to participate in the specimen refinement process, and receive training on the cryo-ET processes involved. Projects with high potential will be referred for high-throughput data collection to the central hub facility of the network. Boulder, Colorado is centrally located and well-connected nationwide through Denver International Airport.