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. 2003 Jan;84(1):571-7.
doi: 10.1016/S0006-3495(03)74877-2.

Variable symmetry in Salmonella typhimurium flagellar motors

Howard S Young  1 , Hongyue Dang, Yimin Lai, David J DeRosier, Shahid Khan
Affiliations

Variable symmetry in Salmonella typhimurium flagellar motors

Howard S Young et al. Biophys J. 2003 Jan.

Abstract

Electron cryomicroscopy of rotor complexes of the Salmonella typhimurium flagellar motor, overproduced in a nonmotile Escherichia coli host, has revealed a variation in subunit symmetry of the cytoplasmic ring (C ring) module. C rings with subunit symmetries ranging from 31 to 38 were found. They formed a Gaussian distribution around a mean between 34 and 35, a similar number to that determined for native C rings. C-ring diameter scaled with the number of subunits, indicating that the elliptical-shaped subunits maintained constant intersubunit spacing. Taken together with evidence that the M ring does not correspondingly increase in size, this finding indicates that rotor assembly does not require strict stoichiometric interactions between the M- and C-ring subunits. Implications for motor function are discussed.

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Figures

FIGURE 1
FIGURE 1
Low magnification view of a field of rotor particles over carbon. Particles in en-face orientation (circled) predominate. Side-on views (boxed) were rare. Bar, 50 nm.
FIGURE 2
FIGURE 2
Side-on views of two rotor particles in ice. These images compare well with side-on views of native rotor particles shown by Thomas et al. (1999, 2001). Vertical bars mark the extent of the C rings. Differences in C-ring diameter between the two particles are apparent. Horizontal bar, 25 nm.
FIGURE 3
FIGURE 3
C-ring subunit symmetry determination for single rotor particles. Each particle was centered with the aid of an annular mask (A). The particle was centered by moving the origin on a 5 × 5 grid, with one pixel separation between grid positions. For each position, the mask was used to compute the rotational cross-correlation function, as described (see Materials and Methods), with the frequency spectrum (B) being obtained by Fourier transformation of the correlation function. (C) Change in amplitude of the peak frequency spectrum as a function of grid position.
FIGURE 4
FIGURE 4
C-ring subunit symmetry changes with particle size. (Left) Different sized particles. (Right) Corresponding composite power spectra. Each composite is composed of 25 superimposed spectra, one for each of the 25 grid positions used for centration. The increase in subunit symmetry with particle size cannot be due to centration error, inasmuch as peak position within each composite spectrum does not change by greater than one.
FIGURE 5
FIGURE 5
Histogram of the number rotor particles as a function of C-ring subunit periodicity. A periodicity of 34–35 subunits was most common. The distribution was well fit by a Gaussian (continuous line). The SD was ±1.4 subunits.
FIGURE 6
FIGURE 6
(A) Average, symmetry-reinforced average, and variance maps of the 34 subunit C-ring rotors (29 images). (B) Cross-correlation function and the resulting power spectrum of the average map. The SD of diameter for particles within this group was ±0.7 nm (i.e., <1 pixel), or 1.7%.
FIGURE 6
FIGURE 6
(A) Average, symmetry-reinforced average, and variance maps of the 34 subunit C-ring rotors (29 images). (B) Cross-correlation function and the resulting power spectrum of the average map. The SD of diameter for particles within this group was ±0.7 nm (i.e., <1 pixel), or 1.7%.
FIGURE 7
FIGURE 7
Symmetry-reinforced, image average maps. The maps are comprised of 9, 12, 29, 38, and 18 particles for the 32, 33, 34, 35, and 36 subunit groups respectively. A Gaussian distribution of subunit symmetries is maintained for this more select dataset. The inner diameter of the 32 subunit C ring, indicated by the red bar, is superimposed on the 34 and 36 subunit C rings to show the progressive increase in diameter with subunit number.
FIGURE 8
FIGURE 8
(A) Evidence for constant intersubunit distance. Diameter of the symmetry-reinforced image averages (closed circles) were plotted as a function of C-ring subunit number. The best-fit line, although constrained to pass through the origin, was imperceptibly affected by the constraint. The regression coefficient is 0.9868 and 0.9866 with and without this constraint, respectively. Its slope yielded an intersubunit spacing of 3.9 nm. Rotor particles with 37 (n = 1) and 38 (n = 3) were also found. The mean diameter values for these are also included (open circles), but were not used for the fit. The mean SD of particle diameter within a symmetry group was ±1.9%. (B) Averaged power spectrum for the 106 particles used to construct the image averages.
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