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

项目摘要 加州大学洛杉矶分校的加州纳米系统研究所(CNSI)的目标是 使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，旨在实现必要的先进成像技术 在纳米尺度上可视化和理解大分子机械的机理。 从之前的NIH S10向EICN提供的资金有助于购买(2006)和安装(2007) 世界上第一台可以工作的泰坦·克里奥斯仪器，它在过去十年里为‘低温EM革命’做出了贡献。 此申请寻求资金购买Glacios，这是一种中级200千伏仪器，具有演示的高电压 分辨率单粒子低温电磁成像能力，架起现有入门级和高端成套产品之间的桥梁 加州大学洛杉矶分校EICN的泰坦·克里奥斯乐器。对这项文书的迫切需要的理由如下： 首先，新仪器将满足广泛范围内对单粒子低温电磁成像的迫切需求 加州大学洛杉矶分校24个实验室由联邦政府资助的生物医学研究项目，包括端粒酶和剪接体 核蛋白复合体；跨膜通道、转运体、杀菌机制和分泌物 系统；与神经退行性疾病有关的复合体。第二，现有的14岁的泰坦·克里奥斯 出现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，另外50%用于满足快速增长的冷冻电子断层扫描需求 加州大学洛杉矶分校(CryoET)用户，得益于其300keV电子和生物量子K3的高穿透能力 成像滤光片。加州大学洛杉矶分校的研究将极大地加强对这种中层低温EM的收购 应用程序中描述的单粒子低温电子显微镜方法测定原子结构。 拟议的文书将得到强有力的机构支持，从而确保产生持久影响。高分辨率 电子成像已经成为CNSI非常成功的研究资源和非常重要的 加州大学洛杉矶分校强大的结构生物学研究社区。数十名活跃的联邦用户的身份识别 资金支持表明，自然科学学院的多个系/研究所都对此很感兴趣。 科学和工程，以及加州大学洛杉矶分校医学院，可能会从获得这一点中受益匪浅 具有已证明的高分辨率低温电磁能力的中级低温电磁仪器。这件乐器，一起 利用现有的微电子衍射(MicroED)、X射线结晶学和核磁共振光谱，将提供一种 为渴望扩展其现有生物医学和技术领域的教师提供强大的资源 包括低温EM在内的生物研究项目。新仪器还将满足对海量数据的迫切需求 为发展低温电子显微镜方法而推动低温电子显微镜发展的装置。不同的生物结构 及其多种多样的体系结构，为科学界提供了丰富的数据源 加州大学洛杉矶分校和普通电子成像社区所追求的方法发展。

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.