High-speed direct detector for cryo electron microscopy

项目摘要 我们正在申请资金，以购买Gatan K3 Summit直接电子探测器，与Titan Krios对接 斯克里普斯研究所的电子显微镜。这一探测器将大大推进 17名NIH资助的研究人员和许多其他研究人员为未来的NIH拨款获得初步数据 应用. Scripps的高效自动化数据采集和实时处理基础设施 使我们的许多教师能够将cryo-EM结构确定方法融入他们的研究中 计划，因此，对cryo-EM设施的需求每年都在继续增加。EM设施 斯克里普斯研究所目前拥有两台电子显微镜，能够产生高分辨率的单 粒子cryo-EM结构-Titan Krios和Talos Arctica，两者都获得了~10岁的图像 Gatan K2 Summit直接探测器。然而，尽管这两种仪器几乎全天候运行， 由于用户需求很高，访问变得越来越有限。目前，用户必须等待十到十二周 在低温EM数据收集会话之间，这必然不利于数十个 研究所内正在进行的NIH资助项目。所要求的K3检测器提高了检测量子 效率，内嵌基于GPU的运动校正，更快的读出速率，以及比现有的更大的视野 K2摄像头。总之，我们估计这些改进将使数据传输速率提高约5.6倍。 在Titan Krios上收集数据，每年能够获得比目前多数千个数据集。 购买这款K3相机还将使我们能够将现有的K2安装在我们的Talos 200 C显微镜上， 进一步提高我们EM设备的效率，使我们的研究人员受益。许多样品，特别是 小于~250千道尔顿的生物目标，无法使用低端CCD相机进行质量评估 我们的其他显微镜。这些初步筛选目前必须使用两个高分辨率 显微镜，因此占用宝贵的时间，否则可以直接对更发达的 项目将当前的K2转移到我们的Talos F200 C将为EM设施提供直接探测器- 使筛选显微镜专用于样品优化，大大提高了我们的研究人员的能力， 确定泰坦Krios和Talos Arctica结构测定的适当条件。斯克里普斯 研究人员长期致力于低温EM技术，研究所的 承诺弥补与购买Gatan K3和四年服务合同有关的短缺 (the其总费用大大超过该补助金的最高预算）。研究所还 承诺拨出资金，以支付每月一次的“发现日”的泰坦Krios，这将提供 研究人员有机会生成初步数据，以寻求NIH资助， 项目通过为我们的调查人员提供必要的显微镜访问，这次升级将有一个 对NIH资助的数十个具有重大生物医学意义的项目产生了变革性影响。

PROJECT ABSTRACT We are requesting funds to acquire a Gatan K3 Summit direct electron detector to interface with the Titan Krios electron microscope at Scripps Research. This detector would significantly advance the research programs of 17 NIH-funded investigators and aid numerous other researchers secure preliminary data for future NIH grant applications. The highly efficient automated data acquisition and real-time processing infrastructure at Scripps has enabled much of our faculty to integrate cryo-EM structure determination methodologies into their research programs, and as a result, the demand on the cryo-EM facility continues to increase every year. The EM facility at Scripps Research currently maintains two electron microscopes capable of generating high-resolution single particle cryo-EM structures - a Titan Krios and an Talos Arctica, both of which acquire images with ~10-year-old Gatan K2 Summit direct detectors. However, despite nearly 24/7 operation of these two instruments, microscope access has become increasingly limited due to high user demand. Currently, users must wait ten to twelve weeks between cryo-EM data collection sessions, which is consequentially detrimental to the progress of dozens of ongoing NIH-funded projects within the Institute. The requested K3 detector has improved detective quantum efficiency, inline GPU-based motion correction, faster read-out rates, and a larger field of view than the existing K2 camera. Together, we estimate that these improvements will result in a ~5.6-fold increase in the rate of data collection on the Titan Krios, enabling acquisition of thousands more datasets per year than currently possible. Acquisition of this K3 camera would also enable us to mount the existing K2 on our Talos 200C microscope, further increasing the efficacy of our EM facility and benefiting our researchers. Many samples, particularly biological targets smaller than ~250 kilodaltons, cannot be assessed for quality using the low-end CCD cameras on our other microscopes. These preliminary screenings currently must be done using the two high-resolution microscopes and consequently occupies precious time that could otherwise be directed towards more developed projects. Transfer of the current K2 to our Talos F200C would provide the EM facility with a direct detector- enabled screening microscope dedicated to sample optimization, dramatically increasing our researchers’ ability to identify appropriate conditions for structure determination on the Titan Krios and Talos Arctica. Scripps Research has a long-standing commitment to cryo-EM technologies, which is demonstrated by the Institute’s commitment to covering the shortfall associated with purchasing the Gatan K3 and four years of service contracts (the total cost of which is significantly more than the maximum budget of this grant). The Institute has also committed to allocating funds to cover a monthly “Discovery Day” on the Titan Krios, which will provide researchers with the opportunity to generate preliminary data to pursue NIH grant funding for exploratory projects. By providing our investigators with the necessary microscope access, this upgrade will have a transformative impact on dozens of NIH-funded projects carrying significant biomedical relevance.