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Figure 1
Data collection setup and SerialEM script. (a) Flowchart outlining steps and the time to set up data collection. A full-grid montage is collected at 62× magnification in lower magnification (LM) mode to identify areas ideal for data collection. Maps of selected grid squares are acquired at 210× magnification in LM mode after finding eucentric height. Multishot record parameters and points are added to each of the grid square maps by microscope operator. (b) Flowchart of the SerialEM script used to collect data. The stage is moved to each of the points (in XYZ) and centered over the hole iteratively using the reference image. Errors in the camera sensor referred to as black strip are checked after hole centering. Autofocus using beam-tilt iterates until target defocus is obtained. Stage drift is measured 20 times, or until drift threshold is obtained. As an option, an additional beam shift is applied to direct the beam away from the center of the hole, we refer to this as touch of carbon and find this useful for particles that cluster close to the edge of the carbon film. Micrographs are acquired in a multishot array, one micrograph is taken at each Quantifoil hole, typically 9–49 holes depending on the hole spacing. (c) Percentage of time spent on each step of the data collection script. Record and delay occur during multishot acquire, with delay including image shift delay, other SerialEM delays (ExtraBeamTime, ShutterDeadTime, ExtraOpenShutterTime), and camera read out and file write time.

IUCrJ
Volume 9| Part 2| March 2022| Pages 243-252
ISSN: 2052-2525
https://doi.org/10.1107/S2052252522000069