CU Boulder Center for Cryo-ET (CCET)

摘要： 我们将根据RFA RM-19-009和-010在更大的网络内建立卫星服务中心， 命名为 CU-Boulder 冷冻电子断层扫描中心 (CCET)。我们的卫星将专注于低温 应用于细胞、细胞器和大型细胞的样本制备和冷冻电子断层扫描 (cryo-ET) 超分子组装体。申请人结合了三位 P.I. 的专业知识，Michael Stowell（高 分辨率冷冻电子显微镜（cryo-EM）），Karolin Luger（大型复杂组件的生物化学， 特别是蛋白质-核酸相互作用），以及 Andreas Hoenger（前 P41 主任，冷冻电镜和冷冻电子断层扫描） 从大分子到细胞和组织）。我们现有的员工将丰富的专业知识与冷冻电子断层技术相结合 以及相关软件。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制备程序以及培训 为那些想要为自己的家建立这些技术或至少其中一部分技术的科学家提供机会 实验室。在博尔德，我们提供多种高端显微镜，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，我们还有全面的 用于冷冻样本制备、存储和处理资源的外围设备，包括计算工具。我们 将添加冷冻聚焦离子束 (cryo-FIB) 铣削以生产细胞和组织的玻璃化薄片。我们建议 该设施的组织如下： 具体目标 1：提供冷冻电子断层扫描 (Cryo-ET) 标本制备方法 服务：我们的用户可以使用我们的技术和专业知识并进行进一步的合作 与并行 RFA (RM-19-010) 中提议的数据收集中心一起。具体目标 2：交叉培训 为服务用户提供机会：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指导。在适当的情况下，我们会将用户分成小组并运行 培训周，作为设备以及我们工厂开发的相关软件的实践研讨会 如串行 EM、IMOD 和 PEET。虽然培训的重点将放在样本的实际方面 在准备和资料收集的过程中，我们经常会遇到新手用户强烈需要彻底介绍的情况 还有图像分析软件。具体目标 3：设施的管理和组织：用户， 通过全网申请流程分配给我们的将有机会参加标本 改进流程，并接受有关冷冻 ET 流程的培训。具有高潜力的项目将 转至网络的中央枢纽设施进行高吞吐量数据收集。科罗拉多州博尔德市位于中心 位于丹佛国际机场，交通便利，连接全国各地。

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.