CU Boulder Center for Cryo-ET (CCET)

预计为2001年提供的所有资金将按计划使用， 预算中详细列出的设备费用预算。 我们将根据RFAs RM-19-009在更大的网络内建立卫星服务中心 和-010，命名为CU-Boulder低温电子断层扫描中心（CCET）。我们 卫星将侧重于低温标本制备和低温电子断层扫描（cryo-ET）应用 到细胞、细胞器和大型超分子组装。申请人将联合收割机的专业知识 三名PI，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）， 和Andreas Hoenger（前P41主任，从大分子到细胞的冷冻EM和冷冻ET 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和相关 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体的制备方法， 以及为希望建立这些技术的科学家提供培训机会，或 至少有一部分是为了他们的家庭实验室在博尔德，我们有几台高端显微镜 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，我们有一个全面的周边冷冻标本 准备、存储和处理资源，包括计算工具。我们会加入冷冻- 聚焦离子束（cryo-FIB）研磨，以生产细胞和组织的玻璃化薄片。我们 提议该设施的组织如下： 具体目标1：为服务提供的Cryo-ET样品制备方法：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）中提议的数据收集中心一起使用。 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，包括一对一的显微镜和相关设备指导。作为 适当的时候，我们将把用户分成小组，并进行一周的培训， 关于设备以及我们工厂开发的相关软件（如Serial-EM）的研讨会， IMOD和PEET。 培训的重点是标本制备和数据的实际方面， 集合，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：通过网络分配给我们的用户 申请过程中将有机会参与标本提炼过程， 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。博尔德， 科罗拉多位于市中心，通过丹佛与全国各地连接良好 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.