Cryo-EM image processing for the rigor complex. (A) Representative cryo-EM micrograph of the human cardiac actin–tropomyosin–myosin rigor complex, taken from a dataset of 3,903. Scale bar: 50 nm. Adjacent to the micrograph are representative 2-D class averages of the complex, where the actin, tropomyosin, and myosin can be seen in more detail. Scale bar: 5 nm. (B) Flowchart describing the cryo-EM data processing scheme to determine the helically averaged human cardiac actin–tropomyosin–myosin rigor complex. This flowchart also includes the focused classification scheme that improved the reconstruction of the myosin–essential light chain complex. Briefly, after generating 2-D class averages from manually picked particles, autopicking produced 1,045,903 segments. Subsequent 2-D and 3-D classifications identified the best segments for use in 3-D auto-refine. Further refinements produced a final helically averaged map to 3.4 Å resolution. Focused classification produced the final 3.8 Å reconstruction of the myosin–essential light-chain complex.