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Fig. 8. Three-dimensional reconstructions of negatively stained thin filaments. Surface views of reconstructions of (A) F-actin control filaments (actin subdomains 1 to 4 marked on one actin monomer) and (B,C) F-actin-Cdc8. Additional density corresponding to tropomyosin (arrows) is observed for the filaments containing Cdc8. Cdc8 occupies the `closed' position on actin, i.e. it localises on the outer edge of actin subdomains 3 and 4 next to the cleft separating the inner (subdomains 3 and 4) and outer (subdomains 1 and 2) domains of actin, which is the same position previously described for troponin-tropomyosin-regulated filaments in the absence of Ca²⁺ (Lehman et al., 1994; Vibert et al., 1997). Reconstructions were displayed at 5 sigma above the mean density in A and B and at 10 sigma in C; note the Cdc8 density forms a continuous strand even at very high threshold values. Helical projections (D,E) and transverse sections (F,G) of maps of the 3D reconstructions in A and B. (D,F) F-actin (E,G), F-actin-Cdc8. Actin subdomains numbered in (F). Helical projections (i.e. densities in the reconstructions projected onto a plane perpendicular to the axis of the filament), which show the average location of Cdc8 (arrows) on the inner domain of actin (E). Transverse sections show that the densities associated with Cdc8 are adjacent to subdomains 2 and 4 of actin (G). Also note that Cdc8 is not observed on pure actin filaments (no Cdc8) (A,D,F).

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