CU Boulder Center for Cryo-ET (CCET)

预计为YR-01年度批出的所有资金将按计划支出，不能重新使用- 对预算中详细说明的设备成本进行预算。 我们将根据RFA RM-19-009在一个更大的网络中建立一个卫星服务中心 和-010，命名为CU-Boulder低温电子断层扫描中心(CCET)。我们的 卫星将专注于低温冷冻剂的制备和冷冻电子断层扫描(Cryo-et)的应用 到细胞、细胞器和大的超分子组件。申请者结合了专业知识 三位私家侦探中，迈克尔·斯托威尔(高分辨率冷冻电子显微镜)，卡罗琳 Luger(大型复杂组装的生物化学，特别是蛋白质-核酸相互作用)， 和安德烈亚斯·霍格(前P41主管，冷冻EM和冷冻-从大分子到细胞 和纸巾)。凭借我们现有的员工，我们将广泛的专业知识与低温技术和相关技术相结合 软件。我们将提供广泛的低温电子显微镜(CRYO-EM)和低温电子显微镜 细胞结构和细胞内大分子组装体的制备程序 以及为希望建立这些技术的科学家提供培训机会，或在 最少的一部分，是为了他们的家庭实验室。在博尔德这里，我们有几台高端显微镜 提供，包括一个新安装的泰坦-克里奥斯。我们的三台显微镜都配备了 直接电子探测器。此外，我们还有全面的冷冻标本外围设备。 准备、存储和处理资源，包括计算工具。我们会加入冰冻- 聚焦离子束(Cryo-FIB)球磨产生细胞和组织的玻璃化片层。我们 建议该设施的组织如下： 具体目标1：提供服务的低温样品制备方法：我们的 我们的用户可以使用技术和专业知识，也可以进行进一步的协作 在平行RFA(RM-19-010)中提议了数据收集中心。 具体目标2：为服务用户提供交叉培训机会：我们将提供全面的 对用户进行培训，包括关于显微镜和相关设备的一对一指导。AS 适当的时候，我们将把用户集中到小组中，并作为实践活动举办培训周 关于我们工厂开发的设备和相关软件的研讨会，如Serial-EM， IMOD和Peet。 虽然培训的重点将放在标本制备和数据的实际方面 收藏时，我们经常会遇到新手用户强烈需要深入介绍 还有图像分析软件。 特定目标 3：设施的管理和组织：用户，通过全网络分配给我们 申请过程将有机会参与样本提炼过程， 并接受有关低温冷冻工艺的培训。具有高潜力的项目将是 用于网络的中央集线器设施的高吞吐量数据收集。博尔德, 科罗拉多州位于中心位置，并通过丹佛在全国范围内建立了良好的联系 国际机场。

All funds awarded for Yr-01 are anticipated to be expended as planned and not available for re- budgeting for the equipment costs detailed in the budget. 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 cryospecimen 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 (highresolution 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 forservice 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.