Rotational symmetry analysis of Dam1C/DASH rings in vivo and in vitro. (A) Left, manually constructed image with perfect 17-fold (outer) and 13-fold (inner) symmetries. The radii of the outer and inner arrays and their aspect ratios are to the approximate scales of a front view of Dam1C/DASH around MTs. Right, rotational power spectrum of the densities on the left. The y axis is the power (arbitrary units), and the x axis is the rotational symmetry. 17-fold symmetry is indicated by the black arrowhead. All subsequent plots have the same axes. (B–G) Left, cryotomographic slices of Dam1C/DASH ring around MTs in vitro, rotated to the front view. Right, power spectra of the cryotomographic slices. Bar, 25 nm for all cryotomographic slices. The non-Dam1C/DASH densities were masked before power spectrum analysis, but the mask is not shown. Some rings, such as those in D and G, are distorted and do not produce a strong peak. The 15-fold symmetry peak comes from the MT densities (many MTs have 15 protofilaments in vitro), which can leak out of the mask due to the missing wedge effect. Note that because these cryotomographic slices were taken coplanar with the Dam1C/DASH ring, the symmetry signal from the MTs is weak or absent when the ring is tilted. (H) Left, cryotomographic slice (6 nm) showing the front view of a Dam1C/DASH ring around a MT in vivo. Right, rotational power spectrum. (I) Subtomogram averages of Dam1C/DASH rings around MTs, without (C1) or with 17-fold (C17) symmetry imposed. The unsymmetrized densities (C1) and subunit numbering are reproduced from Fig. 1 D. Only the most symmetric complexes, corresponding to those that resemble Class 1 in Fig. 1, were symmetrized. The top row is the front view. Each row below is sequentially rotated 45° around the horizontal axis. (J) On the basis of the Fourier-shell correlation = 0.143 criterion, the resolution of the 17-fold symmetrized reconstruction is 32 Å.