Stable interactions between neighboring α1 and α2 helices drive head-to-tail assembly of Vps32 filaments. (A) Two-dimensional class average analysis of vitrified Vps32 polymers. Representative class averages are overlaid to highlight flexibility observed in filaments. The distance between neighboring EM densities is indicated. The red line indicates the separation angle of three density maps with straight orientation. The blue line indicates the separation angle of three density maps with curved orientation. Bars, 5 nm. (B) Docking of Vps32 (top, pseudoatomic model) and human Vps24 (bottom, based on structure 2GD5; obtained from the Protein Data Bank) into an averaged EM isosurface. Helices of each monomer are labeled. (C and D) Homotypic association between Vps32 subunits is predicted to be mediated by their first two α helices. (C) The ZDOCK server was used to determine the relative positions of the helical hairpin from one Vps32 monomer and the asymmetric four-helix bundle of a neighboring Vps32 subunit. (top) The reference four-helix bundle is shown as a ribbon cartoon, and the top 500 predicted positions of the helical hairpin (from an adjacent Vps32 subunit) are shown as gray dots. (bottom) One highly probable position of the helical hairpin (cyan) is shown explicitly. (D) The Vps32 dimer interface is mediated by electrostatic interactions, which arise mainly from the positively charged residues within helices α1′ and α2′ from one monomer (blue) and the negatively charged residues in helices α1 and α2 of a neighboring Vps32 subunit (red). (E) Calculations of the RMSD of the α carbon atoms in the Vps32 dimer interface, relative to a reference structure (left), using an explicit solvent simulation demonstrates its stability. Representative snapshots of the structure at different time points are shown. The interface used for RMSD analysis is colored light red in the reference structure. Interface dynamics at selected time points are reflected by changes in the conformation of overlaid u-shaped lines (red). The black line indicates the RMSD relative to the time of the simulation. Snapshots of the structure at different time points are shown as well. (F) Coomassie-stained fractions of purified, recombinant C. elegans Vps32^E94K after size exclusion chromatography and SDS-PAGE analysis. The monomer peak exhibits a Stokes radius of 3.8 nm. MM, molecular mass. (G) Circular dichroism spectroscopy was used to characterize 1 µM Vps32^E94K. The data shown are from a single experiment, which was repeated twice. (H) Representative images showing the distribution of GFP-CPS in yeast cells lacking endogenous SNF7 and ectopically expressing either wild-type SNF7 (left), an empty vector (middle), or a mutant form of snf7 harboring an E95K substitution (right) from low copy plasmids. (bottom row) Merged images (fluorescence and differential interference contrast [DIC]) are also shown. Bar, 2 µm.