Efficient strategies and troubleshooting for single particle cryoEM data collection using EPU

Abstract Background CryoEM has gained popularity due to advancements in both hardware and software, making it possible to visualize biological molecular structures in detail. However, despite its growing use, cryoEM remains a costly technique, and limited access to cryo-capable TEMs is slowing research efforts. By refining experimental design and streamlining data collection approaches, researchers can significantly boost efficiency and reduce costs, making this technique more accessible and impactful. Method This work presents a guide to some of the session setup parameters for automated cryoEM data collection using Thermo Fisher’s EPU software. Results The TIFF and MRC formats were compared, and the Faster acquisition mode was evaluated against the Accurate acquisition mode. Acquired TIFF files were significantly smaller than MRC files without any notable loss of resolution in the data. By minimizing stage movements, the Faster acquisition mode increases data collection speed, while Accurate acquisition provides precise centering on the optical axis. The models generated from all experiments exhibit similar resolutions, approximately 2.12 Å. These findings suggest that the Faster mode, coupled with recent advancements in hardware and software, can increase data collection speed by nearly 5 times. Discussion This guide offers practical insights into optimizing data collection strategies based on the study's target and sample nature. Such optimization ensures the efficient use of resources, leading to reduced costs and time during both data collection and processing. Based on our findings, we recommend using the Faster mode with counted super-resolution, binning 2, and non-gain normalized TIFF output file format for all strategies outlined below.