A Mid-Level 200kV Instrument for Single-Particle cryoEM

项目摘要 位于洛杉矶的加州大学（UCLA）的加州纳米系统研究所（CNSI）的目标是 是让尖端的纳米生物学和生物医学研究成为可能。作为这一目标的关键部分，电子成像 纳米机器中心（EICN）成立于2007年，以实现必要的先进成像技术 用于在纳米尺度上可视化和理解大分子机械的机制。 从以前的NIH S10到EICN的资金有助于购买（2006）和安装（2007） 世界上第一台工作的Titan Krios仪器，为过去十年的“cryoEM革命”做出了贡献。 该申请寻求资金购买Glacios，这是一种中级200 kV仪器，经证明具有高 分辨率单粒子cryoEM成像能力，以弥合现有的入门级和高端集 泰坦Krios仪器在加州大学洛杉矶分校的EICN。对这一工具的迫切需要的理由如下： 首先，新仪器将满足广泛的单粒子cryoEM成像的迫切需求。 由24个加州大学洛杉矶分校实验室资助的生物医学研究项目，包括端粒酶和剪接体 核蛋白复合物;跨膜通道、转运蛋白、杀菌机制和分泌 系统;和涉及神经退行性疾病的复合物。第二，现有的14岁的泰坦克里奥斯 已经开发了与年龄相关的不稳定问题;此外，其50%的可访问时间是为NIH U24保留的 用户联盟，而另外50%用于满足快速增长的冷冻电子断层扫描的需求 （cryoET）用户在加州大学洛杉矶分校，由于其300 keV电子和BioQuantum K3的高穿透力 成像滤波器。通过收购这种中级cryoEM， 通过本申请中描述的单粒子cryoEM方法进行原子结构测定。 拟议的文书将获得强有力的机构支持，从而确保持久的影响。高分辨率 电子成像已成为CNSI非常成功的研究资源的一个组成部分， 强大的结构生物学研究社区。通过激活的联邦识别数十个用户 资金表明，自然科学学院的多个部门/研究所都有很大的兴趣。 科学和工程，以及加州大学洛杉矶分校医学院，这可能会大大受益于收购这一点， 具有高分辨率cryoEM能力的中级cryoEM仪器。这个工具， 与现有的微电子衍射（microED），X射线晶体学和NMR光谱，将提供一个 强大的资源，为教师谁是渴望扩大其目前的生物医学和 生物研究项目，包括cryoEM。新仪器还将满足对大量数据的关键需求 用于推动cryoID方法开发的cryoEM包络的套件。多样的生物结构 以及它们高度多样的架构，为科学界提供了丰富的数据源， UCLA和一般电子成像社区所追求的方法开发。

Project Summary The goal of the California NanoSystems Institute (CNSI) at the University of California at Los Angeles (UCLA) is to enable cutting-edge nanobiology and biomedical research. As a key part of this goal, the Electron Imaging Center for Nanomachines (EICN) was established in 2007 to enable necessary advanced imaging techniques for visualizing and understanding the mechanisms of macromolecular machineries at the nanometer scale. Funding from a previous NIH S10 to EICN was instrumental to the purchase (2006) and installation (2007) of the world’s first working Titan Krios instrument, which contributed to the ‘cryoEM revolution’ of the last decade. This application seeks funding to purchase a Glacios, a mid-level 200 kV instrument with demonstrated high- resolution single-particle cryoEM imaging capability, to bridge the existing sets of entry-level and high-end Titan Krios instruments at the EICN at UCLA. The critical need for this instrument is justified as follows: First, the new instrument will meet the pressing need for single-particle cryoEM imaging in a broad range of federally funded biomedical research projects by 24 UCLA labs, including telomerase and spliceosomal nucleoprotein complexes; trans-membrane channels, transporters, bactericidal machineries and secretion systems; and complexes involved in neuro-degenerative diseases. Second, the existing 14-year old Titan Krios has developed age-related instability issues; additionally, 50% of its accessible time is reserved for an NIH U24 user consortium, while the other 50% is used to meet the quickly growing need of cryo-electron tomography (cryoET) users at UCLA, thanks to its high penetration power of 300 keV electrons and BioQuantum K3 Imaging filter. UCLA-based research would be greatly enhanced by the acquisition of this mid-level cryoEM for atomic structure determination by the single-particle cryoEM approach described in the application. The proposed instrument will enjoy strong institutional support thus ensuring lasting impact. High-resolution electron imaging has become an integral part of CNSI’s highly successful research resources and of the very strong structural biology research community at UCLA. The identification of tens of users with active federal funding shows that there is major interest across multiple departments/institutes among the colleges of natural sciences and engineering, as well as the UCLA medical school, which may greatly benefit from acquiring this mid-level cryoEM instrument with demonstrated high-resolution cryoEM capability. This instrument, together with existing micro-electron diffraction (microED), X-ray crystallography and NMR spectrometry, will provide a robust resource for faculty members who are eager to expand the scope of their current biomedical and biological research projects to include cryoEM. The new instrument will also meet a critical need for huge data sets for pushing the envelope of cryoEM for the cryoID method development. The diverse biological structures to be studied, and their highly varied architectures, offer a fertile data source for the scientific community for method development pursued by UCLA and the general electron imaging community at large.