ABSTRACT We are requesting funds to acquire a Thermo Fisher Scientific integrated Cryo-Electron Microscopy (Cryo- EM) imaging system (Imaging Energy Filter/Direct Electron Detector) that will upgrade cryo-EM capabilities at the University of Colorado Anschutz School of Medicine Cryo-Electron Microscopy Facility (AnCEM). AnCEM, established in 2016 to enable cryo-EM research on the Anschutz campus and beyond, is backed by strong institutional support from the Anschutz School of Medicine, the Colorado University Cancer Center (UCCC), and the Colorado Clinical and Translational Sciences Institute (CCTSI). The centerpiece of the facility is a Thermo Fisher Scientific 200kV Talos Arctica transmission electron microscope (TEM) equipped with an X-FEG electron source, a phase contrast plate and a Gatan K3 Summit direct electron detector (DED). The facility also counts with a Talos L120C TEM used for cryo-screening and imaging of stained specimens, and with all ancillary equipment required to carry out biomolecular cryo-EM studies. A total of ~50 cryo-EM scientist from ~20 NIH-supported research groups on campus depend on AnCEM for cryo-EM specimen preparation, optimization and screening, and for high-resolution data collection. Over the last 6 years, AnCEM has generated preliminary data for ~20 current NIH-funded grants and generated data included in many peer-reviewed publications. AnCEM’s current imaging capabilities are limited by the lack of an imaging energy filter and by sub-optimal integration of our DED with the Talos Arctica TEM. This has a direct, negative impact on cryo-EM data quality and data collection efficiency, and severely limits our options for selecting automated imaging software on which all data collection depends. These limitations hinder AnCEM’s ability to support cryo-EM research on campus and beyond and compromise the facility’s long-range viability. The proposed imaging system upgrade will allow AnCEM to deliver considerably better cryo-EM data at a much faster rate (effectively reducing data collection time and cost about 3-fold) and allow AnCEM to continue to drive cryo-EM research in Denver and the surrounding area, and to provide essential support to a large number of NIH-funded projects. Regional institutional facilities like AnCEM play an essential role in enabling cryo-EM research, but they must keep up with technological developments to remain relevant and properly fulfill their essential role in getting cryo-EM projects off the ground and complementing data collection resources offered by larger national Cryo-EM facilities. The capabilities of 200kV TEMs have been re-evaluated over the last few years and it is now widely acknowledged that these instruments, which are comparatively cheaper to install and maintain, can be used to determine structures of well-behaved biomolecules at better than 2Å resolution, and to deliver high-quality cryo-electron tomography (Cryo-ET) data. The optics and intrinsic imaging capabilities of our Talos Arctica are comparable to those of the newest 200kV TEMs. Therefore, upgrading the imaging capabilities of our Talos Arctica will address imaging limitations, and dramatically enhance services offered by AnCEM in the most rational and economically-efficient way. The proposed imaging upgrade is essential to support the specific aims of NIH-funded research, and to meet evolving needs of cryo-EM researchers in the CU Anschutz Medical School and beyond. The proposed upgrade will also set the stage for future implementation of large-scale 3D imaging and Cryo-ET on the Anschutz campus.

NARRATIVE The Cryo-EM Facility at the University of Colorado Anschutz School of Medicine is an essential institutional and regional cryo-EM resource. Since it was established in 2016, the facility has supported cutting-edge NIH- funded research from groups across the Medical School and beyond. The centerpiece of the facility is a 200kV Thermo Fisher Talos Arctica transmission electron microscope. The current direct electron detector on the Arctica is 6 years old, starting to become unreliable, and no longer able to generate the best quality data in an efficient and user-friendly manner. Upgrading the imaging system in the Arctica represents the most economical and efficient way to bring the instrument back to the cutting-edge of cryo-EM technology. The imaging upgrade will allow the CU Anschutz Cryo-EM Facility to generate higher quality cryo-EM data in a considerably more efficient way, resulting in major cost benefits, easier data collection, and access to large- scale 3D imaging approaches like cryo-electron tomography. The imaging upgrade will ensure that the facility continues to provide state-of-the-art cryo-EM services for years to come.