The National Cryo-EM Facility
a questionnaire to each user to receive feedback on the data collection process and the quality of the data.
Methods Cryo-EM data collection. Vitrified
Figure 1: Examples of structures solved with the help of data collection at the NCEF. (A) 5-HT3A serotonin
receptor in complex with serotonin [8]. (B) Gasdermin A3 membrane pore [9]. (C) Active Dot1L nucleosome complex [10].
active queue at any one time, and data collection is restricted to 48 hours, unless technical problems with the microscope lead to significant loss (>4 hours) of data collection time.
Data Collection and Transfer Process A typical 48-hour user session starts with transfer of
grids into the microscope and screening of each grid for 10–15 minutes to determine the amount of area that can be imaged and to check the quality of particle projection images. Tis is followed by checking column alignment, including gun and condenser alignment, and collection of a fresh gain reference for the detector. Following these steps, a video conference is initiated with the user to discuss tar- geting and imaging parameters. Once imaging parameters are set, an automated data collection run is set up, and a sufficient number of target areas are selected to sustain approximately 40 hours of data collection. For quality- control purposes, data quality is analyzed concurrently with data collection using Scipion soſtware [1], by MotionCor2 [2] and CTFFind4 [3], and the results are streamed to a web- site that can be accessed by the users. At the end of data collection, the data are com- pressed and transferred to a storage server where they can be downloaded by the users through the Globus GridFTP client. A full report is sent to each user with information on how to download the data. Tis report includes all the imaging settings, comments and recom- mendations on grid and sample quality, and the results from the quality-control analysis. Roughly one month aſter data collection, the NCEF submits
2020 May •
www.microscopy-today.com of 0.85–1.3 Å, a total electron dose between 40–60 e−
grids are imaged using a Titan Krios trans- mission electron microscope (Termo Fisher Scientific) at 300 kV equipped with a K3 direct electron detector and Quan- tum energy filter (both from Gatan Inc.) with a slit width of 20 eV. Data are typi- cally collected with a 100 nm C2 aperture and no objective aperture. Data are usu- ally collected with an unbinned pixel size /Å2
, and
a defocus range of -1.0 to -2.5 μm. Common camera setups use an imaging dose rate of 14–20 e− (2–8 e−
/px/s on the K2 camera). Automated data collection
is performed using Latitude S soſtware from Gatan Inc. To minimize beam tilt distortion, which Latitude S does not cur- rently correct for, image shiſts are set up over short distances of less than 2 μm when multiple images are collected follow- ing the move of the stage to a given position. Te precise set- tings for data collection vary according to user requests and requirements. Determination of ice thickness from a single image.
Electrons passing through a vitreous ice layer may experience either elastic or inelastic scattering. Without an objective aperture, few scattered electrons have scattering angles large
/px/s on the K3 camera
Figure 2: General schematic of the workflow that a user experiences when interacting with the NCEF. 1) The user purifies their protein of interest. 2) The user optimizes freezing conditions for their grids. 3) The user screens grids for quality and sample concentration on a cryo-electron microscope. 4) If screened images are of low quality (damaged grid, thick ice, and/or low sample concentration), the user starts over at either steps 1 or 2 depending on the nature of the problem. 5) If screened grids are of high quality (intact grid, thin ice, and high sample concentration), then the user proceeds with submission. 6) The user fills out a Sample Information Form (SIF) and submits it to the NCEF for review. 7) The SIF is reviewed by NCEF staff, which is either approved within 24 hours or sent back with suggestions on what needs to be done for the submission to be approved. 8) If the sample is approved, the user sends frozen grids to NCEF in an approved dry shipper with grid inventory and return shipment label enclosed. 9) Prior to imaging, NCEF staff makes contact with users to determine imaging parameters, usually via an online video conference. 10) After the imaging session has been completed, the user is able to download their images from the NCEF’s Globus endpoint and receives a detailed report by email.
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