The National Cryo-EM Facility at Frederick National Laboratory: Operational Procedures, Methods, and Outputs
Ulrich Baxa,1 Thomas J. Edwards,1 Joseph Finney,1 Helen Wang,1 *
sriram.subramaniam@
ubc.ca
Abstract: The National Cryo-Electron Microscopy Facility (NCEF) at the National Cancer Institute was launched in May of 2017 to provide free and rapid access to high-resolution cryo-EM data collection to United States researchers working on problems of broad general rel- evance to cancer biology. The decision about suitability of projects for data collection is made on a first-come, first-served basis by NCEF staff and is based solely on the quality of the screening images pro- vided, without need for a scientific proposal. Here we provide an over- view of the operation of the facility, typical data collection procedures, and some insights that have emerged from the structures reported from data collected at the facility.
Keywords: cryo-electron microscopy, electron microscopy facility, ice thickness measurement, cancer
Background Te last few years have seen exponential growth in the
use of cryo-electron microscopy (cryo-EM) by structural biologists. In 2013, only seven cryo-EM density maps were deposited at the Electron Microscopy Data Bank (EMDB,
www.emdatabank.org) at resolutions better than 4 Å, but as of March 2020, over 2000 density maps have been deposited with resolutions better than 4 Å. Te vast majority of these maps have also been of a quality that is good enough to be interpreted in terms of an atomic model of the constituent proteins and related macromolecules. Cryo-EM methods can thus be clearly viewed on par with other experimental approaches, such as X-ray crystallography and nuclear mag- netic resonance spectroscopy, in terms of their potential for atomic-resolution structure determination. Structure determination by cryo-EM involves numerous
experimental and computational steps, but the prohibitive cost of purchasing, maintaining, and operating state-of-the-art microscopes remains a major barrier to using these methods to advance the study of biological mechanisms. Recognizing this need, the National Cancer Institute (NCI) launched the National Cryo-EM Facility (NCEF) in May of 2017 as a feder- ally funded pilot effort to begin meeting the needs of cancer researchers in United States academic labs who do not have adequate access to these instruments. Te goal of the facility is to provide rapid access to users at no cost and with a simplified access model. Our intention in compiling this summary of the NCEF is
three-fold. Te first aim is to present a standardized method for data collection, enabling accurate citation by users when publishing structures based on data generated at the NCEF.
12 doi:10.1017/S1551929520000851
Second, we note observable trends in parameters that appear to be predictive of success in achieving higher resolution based on >350 data collection sessions at the NCEF since its launch (see Figure 1 for some representative structures published using data collected at NCEF). Tird, we present a description of our operational procedures that could poten- tially serve as a reference point for other newly launched cryo-EM user facilities that wish to adopt a similar model for user access.
Access to NCEF NCEF resources are accessible to any United States
researcher from a non-profit organization. Tere is no cost for access, and there is no prerequisite for prior NCI or NIH funding. Users are not required to provide a research proposal but are required to state that their project is cancer-related, which admits a relatively broad scope of projects given that a large number of basic questions in cell biology are relevant to cancer research. Researchers contact the NCEF by sending a submission form (“Sample Information Form” [SIF] available at
https://www.cancer.gov/research/resources/cryoem/access) along with screening images from their grids. If the screen- ing images indicate that the user’s grids are of a quality ade- quate for data collection, the application is approved, typically within 24 hours aſter submission of the application. When the images are deemed to be inadequate to meet standards for data collection at the NCEF, more information might be requested from users, or the NCEF staff will engage with users to help improve sample quality. Once the request is approved, the fro- zen samples are shipped to the NCEF, and the project enters a scheduling queue once samples are received. Te various stages of user engagement with the NCEF are schematically summa- rized in Figure 2. Because samples are not scheduled for a specific date, the
queue at NCEF is dynamic and can adapt rapidly to any delays. Users are encouraged to ship their samples by mail rather than visit in person so that the need for coordination of microscope operations with travel schedules can be minimized. Data runs are set up with the users, directly engaged via video confer- encing. Remote access to a monitoring website, providing on-the-fly feedback on imaging during the run, is available to users and NCEF microscopists. To minimize wait time, only one user project from each principal investigator’s lab is in the
www.microscopy-today.com • 2020 May Matt Hutchison,1 and Sriram Subramaniam1,2
Adam D. Wier,1 *
1Frederick National Laboratory for Cancer Research, Frederick, MD 21701 2University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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