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: 10475160
• 负责人: ANDREAS HOENGER
• 金额: $ 69.95万
• 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/10475160
• 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; 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.

摘要： 我们将根据RFA RM-19-009和-010在更大的网络中建立卫星服务中心， 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- 用于细胞、细胞器和大细胞的样品制备和冷冻电子断层扫描(Cryo-ET) 超分子组装。申请者结合了三位私家侦探的专业知识，迈克尔·斯托威尔(High- 分辨率低温电子显微镜(CRYO-EM))，Karolin Luger(大型复杂组件的生物化学， 尤其是蛋白质-核酸相互作用)，以及Andreas Hoenger(前P41主管，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，我们将广泛的专业知识与低温技术相结合 以及相关的软件。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的准备程序以及培训 为那些想要建立这些技术或至少是其中一部分的科学家提供机会，为他们的家 实验室。在博尔德，我们有几台高端显微镜可供选择，包括一台新安装的泰坦-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，我们还有一个全面的 用于低温样品准备、存储和处理资源的外围设备，包括计算工具。我们 将加入低温聚焦离子束(Cryo-FIB)球磨，以生产细胞和组织的玻璃化薄片。我们建议 该设施的下列组织：具体目标1：提供低温冷冻样品的制备方法 对于服务：我们的技术和专业知识将为我们的用户和进一步的协作提供便利 在平行RFA(RM-19-010)中提议了数据收集中心。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 关于显微镜和相关设备的说明。我们将视情况将用户集中到较小的组中并运行 培训周，作为由我们的设施开发的设备和相关软件的动手研讨会，如 作为Serial-EM、IMOD和Peet。而培训的重点将放在标本的实际方面 在准备和收集数据时，我们经常会遇到新手用户强烈需要深入了解 还有图像分析软件。具体目标3：设施的管理和组织：用户、 通过全网申请程序分配给我们的人将有机会参加样本 精炼流程，并接受有关冷冻-ET流程的培训。具有高潜力的项目将是 用于向网络的中央集线器设施进行高吞吐量数据收集。科罗拉多州的博尔德市位于 通过丹佛国际机场在全国范围内拥有良好的地理位置和网络。