CU Boulder Center for Cryo-ET (CCET)

科罗拉多大学博尔德分校冷冻电子断层扫描中心 (CCET)

• 批准号: 10055681
• 负责人: ANDREAS HOENGER
• 金额: $ 169.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10055681
• 关键词: Area; Biochemistry; Cells; Cellular Structures; Collaborations; Colorado; Communities; Complex; Computer software; Cryo-electron tomography; Cryoelectron Microscopy; Crystallography; Data Analyses; Data Collection; Educational workshop; Electron Microscopy; Electrons; Equipment; Fibroblasts; Fluorescence Microscopy; Fostering; Freeze Substitution; Freezing; Funding; Home environment; Image; Image Analysis; Instruction; International; Ions; Knowledge; Light; Metallothionein; Metals; Methods; Microscope; Microtome - medical device; Microtomy; Microtubules; Morphologic artifacts; Names; Nucleic Acids; Organelles; Organization and Administration; Participant; Plastic Embedding; Preparation; Procedures; Process; Proteins; Protocols documentation; Questionnaires; Resolution; Resources; Roentgen Rays; Route; Running; Sampling; Scientist; Series; Services; Specimen; Students; Synchrotrons; Technology; Temperature; Testing; Thick; Thinness; Time; Tissues; Titan; Training; Virus; Work; base; computerized tools; detector; electron density; expectation; experience; instructor; light microscopy; macromolecular assembly; macromolecule; particle; pressure; software development; technology training; training opportunity; web site

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.

概括： 我们将根据RFAS RM-19-009和-010在较大网络中建立一个卫星服务中心， 被命名为Cu-Boulder冷冻电子断层扫描中心（CCET）。我们的卫星将专注于冷冻 标本制备和冷冻电子断层扫描（冷冻-ET）应用于细胞，细胞器和大型 超分子组件。申请人结合了三个P.I.S的专业知识，Michael Stowell（高级 分辨率的低温电子显微镜（Cryo-EM）），Karolin Luger（大型复合物组件的生物化学， 特别是蛋白质核酸相互作用）和Andreas Hoenger（前P41导演Cryo-EM和Cryo-ET 从大分子到细胞和组织）。与当前的员工一起，我们将广泛的专业知识与Cryo-Et结合在一起 和相关软件。我们将提供广泛的低温电子显微镜（Cryo-EM）和Cryo-ET 细胞结构和细胞内大分子组件的制备程序，以及训练 希望建立这些技术或至少一部分为他们的家的科学家的机会 实验室。在Boulder，我们有几个高端显微镜，包括新安装的Titan-Krios。 我们的三个显微镜配备了直接电子探测器。此外，我们还有一个全面的 用于冷冻特性准备，存储和处理资源（包括计算工具）的外围设备。我们 将添加以低温为中心的离子束（冷冻纤维）铣削，以产生细胞和组织的玻璃薄片。我们建议 设施的以下组织：特定目的1：CRYO-ET标本准备方法提供的方法 服务：我们的技术和专业知识将为我们的用户访问并进行进一步的协作 在平行RFA（RM-19-010）中提出的数据收集中心。特定目标2：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 有关显微镜和相关设备的说明。我们将在适当的情况下将用户列入小组并运行 作为设备的动手研讨会以及我们设施开发的相关软件的培训周 作为串行EM，IMOD和PEET。而培训的重点将放在标本的实际方面 准备和数据收集，我们经常遇到新手用户，非常需要彻底介绍 图像分析软件。特定目标3：设施的管理和组织：用户， 通过网络范围的应用程序分配给我们将有机会参与标本 改进过程，并接受有关涉及的冷冻过程的培训。潜力很高的项目将是 将高通量数据收集转介给网络的中央集线器设施。科罗拉多州博尔德市中心 位于丹佛国际机场的全国性位置和连接良好。