Cryo‐EM map interpretation and protein model‐building using iterative map segmentation

A procedure for building protein chains into maps produced by single‐particle electron cryo‐microscopy (cryo‐EM) is described. The procedure is similar to the way an experienced structural biologist might analyze a map, focusing first on secondary structure elements such as helices and sheets, then varying the contour level to identify connections between these elements. Since the high density in a map typically follows the main‐chain of the protein, the main‐chain connection between secondary structure elements can often be identified as the unbranched path between them with the highest minimum value along the path. This chain‐tracing procedure is then combined with finding side‐chain positions based on the presence of density extending away from the main path of the chain, allowing generation of a C(α) model. The C(α) model is converted to an all‐atom model and is refined against the map. We show that this procedure is as effective as other existing methods for interpretation of cryo‐EM maps and that it is considerably faster and produces models with fewer chain breaks than our previous methods that were based on approaches developed for crystallographic maps.