Figure 2.

(A) Diagram outlining the first three phases of single particle analysis in Bsoft. The preprocessing of the micrographs can be done as the data is acquired on the microscope, including determining CTF parameters. The CTF parameters are then checked manually, the particle template produced, and particles picked. An initial, low‐resolution reference map is produced and the particles aligned and reconstructed iteratively until the resolution stops improving. (B) For validation by independent sets, the micrographs are split into two subsets and processed separately. The two maps are then compared by Fourier shell correlation to assess the resolution and whether the processing is diverging. (C) The final phase, interpretation, requires extensive case‐specific input from the user for identification and annotation of components, segmentation into regions‐of‐interest, quantitation of parts, and building detailed or coarse‐grained models