Figure 4. Cyclical workflow of iterative density sharpening using reference-based scaling and successive completion of atomic models.

(A) An atomic model is used as a reference to improve contrast in the original cryo-EM reconstruction employing local amplitude scaling (1). The resulting LocScale map contains improved contrast and thus facilitates further model building (2) and coordinate refinement (3). Scaling, building and refinement steps can be iterated to produce successively improved density maps and atomic models. (B) Workflow for extending atomic model beyond the reference structure using the LocScale procedure. (Top left) EM density for TRPV1 (EMD-5778) superposed onto an incomplete atomic model covering only the transmembrane region. In three successive cycles of LocScale sharpening and extension of the initial coordinate set by model building into new density with improved contrast, the atomic model can be completed to interpret the entire experimental density from EMD-5778. An effective window size of 25 Å was used for the LocScale iterations.