Fig. 6. Ultrastructure of negatively stained crescentin mutants. Samples of purified crescentin mutants (0.2 mg/mL, 4 lM) are in 50 mM PIPES (pH 6.5), HEPES (pH 7.5), or Tris-HCl (pH 8.5) unless otherwise noted. Kþ and Mg2þ denote 200 mM KCl and 5 mM MgCl2, respectively. (a) CrescentinDL1 in 5 mM buffer. Single 9-nm-wide filaments are denoted with arrows, and filaments with approximate widths of 18 nm (arrowhead) or 27 nm (asterisk) are marked. (b) CrescentinDL1 in 50 mM buffer. The arrowhead denotes an 11 nm-wide fil- ament. (c) CrescentinDL1 in the presence of 200 mM Kþ. Arrowheads mark globular structures with approximate diameters of 20–30 nm. (d) CrescentinDL1 in the presence of 5 mM Mg2þ. The arrow marks a 50 nm-wide structure. (e) CrescentinDL1 at pH 7.5 with 5 mM Mg2þ. A structure with 80 nm width is marked (arrow). (f ) CrescentinDN27 in 50 mM buffer. Arrows denote 8–9 nm-wide filaments (also in inset), the arrowhead denotes a 16 nm-wide filament, and the asterisk denotes a 24 nm-wide filament. (g) CrescentinDN27 in the presence of 5 mM Mg2þ. Arrow in inset denotes a 9 nm-wide filament, and the arrowhead denotes a 17 nm-wide filament. (h) CrescentinDN27 in the presence of 200 mM Kþ. Arrow denotes a 22 nm-wide filament. (i) CrescentinSAT. A single 9 nm-wide filament (inset, arrowhead) and 40 nm-wide bundles (arrows) are denoted. Bundled areas showed an axial repeat of 57.1 6 0.7 nm (n ¼ 25). (j) CrescentinSAT in the presence of 5 mM Mg2þ. The arrowhead marks a 10 nm-wide filament. (k) CrescentinSAT at pH 7.5 in the presence of 5 mM Mg2þ. The paracrystalline struc- tures have variable width and an axial repeat of 58.7 6 0.3 nm (n ¼ 30). Inset shows a detail of the striations. (l) CrescentinDT. Single fila- ments (arrowheads, inset) displayed widths of 17–20 nm. (m) CrescentinDT in the presence of 5 mM Mg2þ. Tapered paracrystalline bundles had an axial repeat of 55.6 6 0.6 nm (n ¼ 30). (n) CrescentinDT at pH 7.5 in the presence of 5 mM Mg2þ.