Acquisition of a 300 kV Electron Microscope for Structural Biology

 描述（由申请人提供）：我们建议获得FEI Krios 300 kV透射电子冷冻显微镜，该显微镜配备计数模式电子检测器、相位板保持器、Gatan柱后能量过滤器（带K2-XP相机）和自动数据采集软件。该设施将由国家高分子成像中心（NCMI）管理，该中心是贝勒医学院的NIGMS支持的生物医学技术资源中心。除了技术开发之外，NCMI的主要任务之一是与需要冷冻电子显微镜（CryoEM）和冷冻电子断层扫描（CryoET）的研究人员合作并提供服务，以便以原子到纳米的分辨率确定从大分子到细胞的结构。我们在本提案中确定了7个主要用户和10个次要用户，他们已经是休斯顿地区内外的NCMI活跃用户。该仪器设施是针对我们的用户谁有一个或多个项目与以下目标：（1）原子分辨率结构测定分子机器，（2）单粒子结构与构象和/或组成异质性，（3）亚细胞和大分子研究细胞在正常和病理状态。所有这些用户的项目都需要高通量和高质量的数据，这些数据将导致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的数据，我们将以类似于X射线同步加速器光束线的方式管理我们的设施，以提供每天24小时和每周6天（24/6）的运行。现有的NCMI电子显微镜都无法提供与CryoEM/ET项目当前技术水平所需的高通量相当的数据质量。实际的显微镜调度将根据用户的偏好和显微镜的可用性每季度进行一次。拟议的仪器将由两名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帮助下收集数据。我们计划在运作的头18个月后向NCMI的其他用户提供拟议的仪器。然后，由PI任命的CryoEM/ET专家组成的外部用户项目评估委员会将对现有和新用户提交的提案进行评估和排名。当局会成立两个谘询委员会，监察拟议文书的运作和成效。贝勒医学院研究高级副总裁将任命一个当地委员会;而PI将根据NIGMS支持的P41中心的授权成立一个外部委员会。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。我们通过现有的P41中心拨款资助NCMI，为这一拟议工具的运作提供了足够的NIH支持，该基金现已进入第30个年头，至少延长至2020年1月1日。我们预计将寻求竞争性更新该补助金，以在2020年以后继续运营拟议的设施。如果这被证明是不成功的，我们计划将拟议的仪器成为贝勒医学院先进技术核心实验室（ATCL）管理下的核心实验室，该实验室将提供仪器运营成本（服务合同，人员和用品）的一半，并使用已建立的ATCL成本充值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.