Heterogeneity in the reconstruction. 3-D classification using RELION produced six classes of conformation (A–F, classes 1–6, respectively), showing that the 10S myosin molecules are quite heterogeneous, due either to variable staining and/or other factors during specimen preparation or to natural variation in the molecules themselves. (A) The tail density partially disappeared in some regions (red dotted circles; cf. red arrow in C, where it is visible). (B) The tail cannot be explicitly identified in this reconstruction, and the second hinge point (star in C) is missing (blue circle). The reconstructions in C and D showed similar structural features, with both the interacting heads and the tail clearly visible. (E) Segment 2 around the blocked head is not visible, and the second hinge point is absent (blue circle), as in B. (F) The tail wraps around the blocked head, but the density present in C (cyan arrow) and D (cyan circle) is absent.