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Methods Enzymol. 2016;579:191-226. doi: 10.1016/bs.mie.2016.04.013. Epub 2016 Jun 7.

Frealign: An Exploratory Tool for Single-Particle Cryo-EM.

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Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States. Electronic address:


Frealign is a software tool designed to process electron microscope images of single molecules and complexes to obtain reconstructions at the highest possible resolution. It provides a number of refinement parameters and options that allow users to tune their refinement to achieve specific goals, such as masking to classify selected regions within a particle, control over the refinement of specific alignment parameters to accommodate various data collection schemes, refinement of pseudosymmetric particles, and generation of initial maps. This chapter provides a general overview of Frealign functions and a more detailed guide to using Frealign in typical scenarios.


Asymmetry; Classification; Contrast transfer function; High resolution; Masking; Refinement

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