Spiral assembly of Vps32 filaments harnesses intrinsic free energy. (A) Two views showing the bending flexibility of a Vps32 monomer (left) and a human Vps24 monomer (right) in MD simulations. In both cases, bending of the helical hairpin is restricted to a single plane. (B) In silico free energy analysis of Vps32 at different bending angles. The bending angle is defined in Fig. 6 B and normalized to 0°, as is observed in the pseudoatomic structure of Vps32 and the crystal structure of human Vps24. (right) A model showing the changes in free energy throughout the spiral is provided. (C) Bending angle analysis of a vitrified Vps32 spiral pair. The filament trajectory is labeled (red, black, and blue) and plotted (right). Linear treadlines that were fitted to each portion of the graph are shown. The termini of the spiral pair are indicated (S [starting point] and E [ending point]). Bar, 25 nm. The data shown are from a single representative experiment, which was repeated three times (n = 15 for the experiment shown). (D) Summary of bending angles within spirals (n = 25 spiral pairs). Error bars represent means ± SEM.