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Image collection strategies for single particle cryoEM

Advancements in direct electron detectors, such as larger sensors and higher refresh rates, as well as data processing software, have contributed to the continuous increase in the number and resolution of cryoEM structures [1]. Underpinning the explosion in new structures are image collection strategies whereby electron beam tilt is implemented to acquire images from holes in a ~5 μm radius from the center of the cluster without moving the stage. Understanding of the effects of beam-tilt on image quality [2], implementation of beam tilt compensation in RELION [3] and calibration of comma versus image shift implemented in SerialEM [4], have together led to a significant increase in image acquisition rates in the past few years. Here, we discuss some parameters that users can consider when deciding on the optimal data collection strategies and magnifications. While the concepts used in this work can be generalized to other detectors and electron microscopes, the estimates presented here are for the 300 kV Titan Krios microscopes coupled with Gatan K3 direct electron detectors.