Acquisition of a 300 kV Electron Microscope for Structural Biology

 描述(申请人提供)：我们建议购买一台FEI Krios 300千伏透射式电子冷冻显微镜，配备一个计数模式电子探测器、一个相位板支架、一个带有K2-XP相机的Gatan柱后能量过滤器和自动数据采集软件。该设施将由国家大分子成像中心(NCMI)管理，NCMI是贝勒医学院由NIGMS支持的生物医学技术资源中心。除了技术开发，NCMI的主要任务之一是与需要低温电子显微镜(Cryo-Electronics显微镜，CryoEM)和低温电子断层扫描(Cryo-Electronics Tomography，CryoET)以原子到纳米分辨率确定从大分子到细胞的结构的研究人员合作，并为他们提供服务。我们在该提案中确定了7个主要用户和10个次要用户，他们在休斯顿地区内外都已经是活跃的NCMI用户。该仪器设备面向具有以下一个或多个目标的项目的用户：(1)分子机器的原子分辨率结构测定；(2)具有构象和/或组成异质性的单颗粒结构；(3)正常和病理状态下细胞的亚细胞和大分子研究。所有这些用户的项目都需要高通量和高质量的数据，这将导致3D结构产生与癌症、传染病和神经疾病相关的分子和细胞成分以及生物过程的新结构见解。由于每个项目都需要大量数据，我们将管理我们的设施，类似于X射线同步加速器光束线，提供每天24小时、每周6天(24/6)的操作。现有的NCMI电子显微镜都不能提供与当前最先进的CryoEM/ET项目所需的高通量相媲美的数据质量。实际的显微镜调度将根据用户的喜好和显微镜的可用性按季度进行。拟议的文书将由两名NCMI工作人员维护和操作，他们将协助用户收集数据。我们将为用户及其实验室成员提供定期培训课程，使他们能够在我们工作人员的最低限度协助下收集数据。我们计划在NCMI的头18个月后向其他NCMI用户提供建议的工具。然后，由国际和平研究所任命的由CryoEM/ET专家组成的外部用户项目评估委员会将对现有用户和新用户提交的提案进行评估和排名。将设立两个咨询委员会，以监督拟议文书的运作和生产力。一个地方委员会将由贝勒医学院负责研究的高级副总裁任命；而根据NIGMS支持的P41中心的授权，PI将设立一个外部委员会。该仪器的总成本约为600万美元。贝勒医学院承诺提供400万美元的配套资金，用于购买仪器和实验室翻新费用。通过现有的P41中心拨款资助NCMI，我们得到了NIH对这一拟议工具的足够支持，目前NCMI已进入第30个年头，至少延长到2020年1月1日。我们预计将寻求这笔赠款的竞争性续期，以便在2020年后继续运营拟议的设施。如果不成功，我们计划将拟议的仪器变成贝勒医学院先进技术核心实验室(ATCL)管理下的核心实验室，该实验室将提供一半的仪器运营成本(服务合同、人员和用品)，并使用现有的ATCL成本补充机制来收回剩余的运营成本。

 DESCRIPTION (provided by applicant): We propose to acquire a FEI Krios 300 kV transmission electron cryo-microscope equipped with a counting mode electron detector, a phase plate holder, a Gatan post-column energy filter with a K2-XP camera and automated data acquisition software. This facility will be administered by the National Center for Macromolecular Imaging (NCMI), a NIGMS supported Biomedical Technology Resource Center at Baylor College of Medicine. In addition to technology development, one of the NCMI's primary missions is to collaborate with and provide service to researchers who need Cryo-electron microscopy (CryoEM) and Cryo-electron tomography (CryoET) for structure determinations from macromolecules to cells at atomic to nanometer resolutions. We have identified 7 major users and 10 minor users in this proposal who are already active NCMI users both in and outside the Houston Area. This instrument facility is aimed for our users who have projects with one or more of the following objectives: (1) atomic resolution structure determination of molecular machines, (2) single particles structures with conformational and/or compositional heterogeneity, (3) subcellular and macromolecular studies of cells in normal and pathological states. All of these users' projects require high throughput and high quality data which will lead to 3-D structures yielding new structural insights of molecular and cellular components and biological processes related to cancer, infectious and neurological diseases. Due to the large quantity of data needed for each of these projects, we will manage our facility similar to an X-ray synchrotron beamline to provide 24 hours per day and 6 days per week (24/6) operation. None of the existing NCMI electron microscopes can deliver comparable data quality with the high throughput needed by the current state of art of CryoEM/ET projects. The actual microscope scheduling will be made quarterly by considering users' preference and the microscope availability. The proposed instrument will be maintained and operated by two NCMI staff who will assist users with data collection. We will provide regular training courses to users and their lab members to enable them to collect data with minimal assistance from our staff. We plan to make the proposed instrument available to other NCMI users after the first 18 months of operation. Then, an external user project evaluation committee made of CryoEM/ET experts appointed by the PI will evaluate and rank the proposals submitted by existing and new users. Two advisory committees will be set up to monitor the operation and productivity of the proposed instrument. One local committee will be appointed by the Senior Vice President of Research at Baylor College of Medicine; while an external committee is set up by the PI as mandated by the NIGMS supported P41 Center. The total cost of the instrument is ~$6 million. Baylor College of Medicine is committed to provide matching funds of $4 million towards the purchase of the instrument and the cost for the laboratory renovation. We have the adequate NIH support for the operation of this proposed instrument via the existing P41 Center grant funding the NCMI, now in its 30th year, extending at least until January 1, 2020. We expect to seek competitive renewal of this grant to continue the operation of the proposed facility beyond 2020. Should this prove unsuccessful, we plan for the proposed instrument to become a Core Lab under the management of the Advanced Technology Core Laboratories (ATCL) at Baylor College of Medicine, which will provide half of the instrument operational costs (service contract, personnel and supplies) and use established ATCL cost recharge mechanisms to recover the remaining operational costs.