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

Ali Punjani; Haowei Zhang; David J. Fleet

The PfCRT-Fab complex (100kDa) is especially interesting due to its small size on the spectrum of proteins solvable by cryo-EM. In addition, the lipid nanodisc (∼50kDa) around PfCRT accounts for a large fraction of the total particle molecular weight. It is precisely in cases like this that the nonuniform refinement strategy is critical, as larger disordered regions leave more room for over-and underregularization to influence particle alignments. For PfCRT in particluar, Fig. 4B shows substantial changes in particle alignments between uniform and non-uniform refinement. Most particle images exhibit pose changes of more than 6 degrees, indicating that regularization has a large impact on alignment. It also indicates that a large fraction of particle images may have been grossly misaligned by uniform refinement. Figure 4D shows in detail the quality of the non-uniform refinement map for PfCRT. Transmembrane α-helices can be directly traced, including side-chains. In contrast, the uniform refinement map does not show helical pitch, and cannot separate β-strands in the Fab domain.