High-speed direct detector for cryo electron microscopy

项目摘要 我们正在申请资金购买Gatan K3 Summit直接电子探测器，以便与泰坦Krios对接 斯克里普斯研究中心的电子显微镜。这一探测器将极大地推进 17名NIH资助的研究人员和众多其他研究人员获得了未来NIH拨款的初步数据 申请。斯克里普斯高效的自动化数据采集和实时处理基础设施 使我们的许多教员能够将低温EM结构确定方法整合到他们的研究中 因此，对低温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维护着两台电子显微镜，能够产生高分辨率的单个 粒子冷冻-EM结构-泰坦克里奥斯和塔洛斯北极，这两个都获得了~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，显微镜 由于高用户需求，访问变得越来越有限。目前，用户必须等待10到12周 在冷冻-EM数据收集会议之间，这对数十个 研究所内正在进行的由国家卫生研究院资助的项目。请求的K3探测器提高了探测量子 更高的效率、基于GPU的内联式运动校正、更快的读出速度和比现有的更大的视场 K2相机。我们估计，这些改进加在一起将使数据率提高约5.6倍 在泰坦Krios上收集数据，使每年能够比目前可能获得的数据集多数千个。 收购这款K3相机还将使我们能够将现有的K2安装在我们的Talos 200C显微镜上， 进一步提高我们的EM设施的效率，并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 在我们的其他显微镜上。这些初步筛选目前必须使用两个高分辨率 显微镜，因此占用了宝贵的时间，否则可以用来进行更多的开发 项目。将当前的K2转移到我们的Talos F200C将为EM设施提供一个直接探测器- 启用筛选显微镜，致力于样品优化，极大地提高了我们的研究人员的能力 为确定土卫六、克里奥斯和塔洛斯极地的结构确定合适的条件。斯克里普斯 研究人员长期致力于低温电磁技术，这一点从该研究所的 承诺弥补与购买Gatan K3和四年服务合同相关的缺口 (其总费用远远超过这笔赠款的最高预算)。该研究所还拥有 承诺拨出资金用于在泰坦克里奥斯上举办每月一次的“探索日”活动，这将提供 研究人员有机会生成初步数据，以寻求NIH探索性拨款 项目。通过为我们的调查人员提供必要的显微镜访问，此次升级将具有 对美国国立卫生研究院资助的数十个具有重大生物医学意义的项目产生变革性影响。

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.