Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

Ali Punjani; Haowei Zhang; David J. Fleet

Compared to the preceding datasets, the Na v 1.7 complex has a larger molecular weight, and in relative terms, a smaller detergent micelle. It does, however, have other regions that are disordered or flexible, namely, a central 4-helix bundle, peripheral transmembrane domains, and the Fabs. Notably, non-uniform refinement handles these regions of the structure automatically along with the micelle, and therefore still delivers improved resolution and map quality. Figure 6A shows FSC curves from uniform and nonuniform refinement. Uniform refinement already reaches a 3.4Å resolution, and non-uniform refinement further improves this to 3.1Å. Figure 6C shows 3D density maps colored by local resolution. With nonuniform refinement, map quality is clearly improved in central transmembrane regions, while flexible parts of the structure (membrane α-helices closest to the micelle, Fab domains, and 4-helix bundle) remain at intermediate resolutions. Figure 6B shows changes in particle alignment poses and shifts between the two refinement types. These changes are smaller on average than the other examples, congruent with the smaller changes in map features. Figure 6D shows three selected regions from the density maps along with a docked atomic model (PDB-6N4Q [33] ). In α-helices farther from the center of the protein (left, center), improvement in map quality is readily apparent, with many side chains being somewhat buildable in the uniform refinement map, but having clearly interpretable density in the non-uniform refinement map. Central α-helices (right) show less improvement, but map quality remains equal or slightly improved. This result is significant because it shows that reconstructions of proteins without disorder are not harmed by using non-uniform refinement.