A systematic and quantitative evaluation of cryo-EM maps is necessary to judge their quality and to capture all possible sources of error. A single value for global resolution is insufficient to accurately describe the quality of a reconstructed density. We describe the estimation and evaluation of two additional resolution measures, local and directional resolution, using methods based on the Fourier shell correlation (FSC). We apply the protocol to samples that encompass different types of pathologies a user is expected to encounter and provide analyses on how to interpret the output files and resulting maps. Implementation of these tools will facilitate density interpretation and can guide the user in adapting their experiments to improve the quality of cryo-EM maps, and by extension atomic models.
Keywords: Anisotropy; Cryo-EM; Fourier shell correlation; Preferred orientation; Sampling; Single-particle analysis.