(A) 90° light scattering assay of crenactin polymerisation. Arrow indicates ATP addition. Crenactin polymerisation is shown in dark blue (positive control). Curves representing the depolymerisation of crenactin by addition of arcadin-2 and arcadin-2 C-terminal peptide (residues 187–203) are shown in red and green, respectively. A curve following the addition of arcadin2ΔC (residues 1–167, only) is shown in purple. Crenactin and arcadin-2 premixed before the experiment is shown with the light blue curve. (B) Analytical ultracentrifugation profile of crenactin and crenactin with arcadin-2 C-terminal peptide (residues 187–203), showing monomers only for the complex sample. (C) Size exclusion chromatography profile of the crenactin:arcadin-2 complex, with corresponding Coomassie-stained SDS-PAGE. (D) Ribbon/surface representation of crenactin:arcadin-2 peptide (residues 187–203) complex crystal structure at 1.6 Å resolution, showing the binding of arcadin-2 to the hydrophobic groove, where the D-loop binds in filaments of crenactin. (E) Ribbon representation of two subunits of crenactin in the filament. The localisation of the arcadin-2 C-terminal peptide (187–203) is shown in black. Note the clash between the presence of the arcadin-2 peptide and the polymer form of crenactin, especially the D-loop. (F) Ribbon representation of archaeal, eukaryotic and bacterial actins in complex with protein domains involved in the regulation of the filaments. PDB IDs: crenactin:arcadin-2 5LY3 (this work); actin:thymosin β4 4PL8 (Xue et al., 2014); ParM:ParR 4A62 (Gayathri et al., 2012). Note that the orientation of the thymosin peptide is reversed in comparison with arcadin-2 and ParR.