Fig 6.

Ultrastructure of negatively stained crescentin mutants. Samples of purified crescentin mutants (0.2 mg/mL, ∼ 4 μM) are in 50 mM PIPES (pH 6.5), HEPES (pH 7.5), or Tris-HCl (pH 8.5) unless otherwise noted. K⁺ and Mg²⁺ denote 200 mM KCl and 5 mM MgCl₂, respectively. (a) Crescentin_ΔL1 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) Crescentin_ΔL1 in 50 mM buffer. The arrowhead denotes an 11 nm-wide filament. (c) Crescentin_ΔL1 in the presence of 200 mM K⁺. Arrowheads mark globular structures with approximate diameters of 20–30 nm. (d) Crescentin_ΔL1 in the presence of 5 mM Mg²⁺. The arrow marks a 50 nm-wide structure. (e) Crescentin_ΔL1 at pH 7.5 with 5 mM Mg²⁺. A structure with 80 nm width is marked (arrow). (f) Crescentin_ΔN27 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) Crescentin_ΔN27 in the presence of 5 mM Mg²⁺. Arrow in inset denotes a 9 nm-wide filament, and the arrowhead denotes a 17 nm-wide filament. (h) Crescentin_ΔN27 in the presence of 200 mM K⁺. Arrow denotes a 22 nm-wide filament. (i) Crescentin_SAT. 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 ± 0.7 nm (n = 25). (j) Crescentin_SAT in the presence of 5 mM Mg²⁺. The arrowhead marks a 10 nm-wide filament. (k) Crescentin_SAT at pH 7.5 in the presence of 5 mM Mg²⁺. The paracrystalline structures have variable width and an axial repeat of 58.7 ± 0.3 nm (n = 30). Inset shows a detail of the striations. (l) Crescentin_ΔT. Single filaments (arrowheads, inset) displayed widths of 17–20 nm. (m) Crescentin_ΔT in the presence of 5 mM Mg²⁺. Tapered paracrystalline bundles had an axial repeat of 55.6 ± 0.6 nm (n = 30). (n) Crescentin_ΔT at pH 7.5 in the presence of 5 mM Mg²⁺.