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. 2006 Oct;188(20):7039-48.
doi: 10.1128/JB.00552-06.

The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium

Dennis R Thomas  1 , Noreen R Francis, Chen Xu, David J DeRosier
Affiliations

The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium

Dennis R Thomas et al. J Bacteriol. 2006 Oct.

Abstract

Three-dimensional reconstructions from electron cryomicrographs of the rotor of the flagellar motor reveal that the symmetry of individual M rings varies from 24-fold to 26-fold while that of the C rings, containing the two motor/switch proteins FliM and FliN, varies from 32-fold to 36-fold, with no apparent correlation between the symmetries of the two rings. Results from other studies provided evidence that, in addition to the transmembrane protein FliF, at least some part of the third motor/switch protein, FliG, contributes to a thickening on the face of the M ring, but there was no evidence as to whether or not any portion of FliG also contributes to the C ring. Of the four morphological features in the cross section of the C ring, the feature closest to the M ring is not present with the rotational symmetry of the rest of the C ring, but instead it has the symmetry of the M ring. We suggest that this inner feature arises from a domain of FliG. We present a hypothetical docking in which the C-terminal motor domain of FliG lies in the C ring, where it can interact intimately with FliM.

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Figures

FIG. 1.
FIG. 1.
Schematic drawing of the hook and basal body. At the tip of the HBB are the three hook-associated proteins. The hook extends from them to the rod. The rod passes through the L and P rings, which form a bushing through the lipopolysaccharide and the peptidoglycan layers. It stops just short of the M ring, which is embedded in the plasma membrane. The M ring passes through the membrane and extends into the cytoplasm. The C ring lies beneath the M ring held by thin connections sometimes seen in images of HBBs.
FIG. 2.
FIG. 2.
Data used to determine the best method for correction of the contrast transfer function. (a) Plots of rotationally averaged Fourier amplitude versus radius. Individual micrographs were divided into a set of segments having dimensions of 128 by 128, 256 by 256, or 512 by 512. The power spectra from each of the segments were averaged together, and the result was rotationally averaged. A background subtraction is applied to the power spectra as part of the program CTFFIND2 (29). Note that the 512-by-512 area produced the best result. (b) Plots showing the effect of the choice of noise-to-signal ratio on the corrected power spectrum (see Materials and Methods). When the value is too small, minima are introduced into what should be peaks (arrow), and bifurcated peaks are produced where there should be a node (arrowhead). At a signal-to-noise ratio of 0.5, this artifact disappears. The power spectra have been calculated using SPIDER (13) and have not had any background subtracted. (c) Comparison of the original CTF determined in a 512-by-512 box with the CTF-corrected power spectrum of the whole micrograph. When the whole micrograph is corrected, note how well the positions of the nodes are preserved and how close the nodes are to zero.
FIG. 3.
FIG. 3.
Steps in generating a reference for image sorting and alignment. (a) The rotationally averaged, 34-fold top view of a C ring. (b) Side view of the C ring, which is similar to that shown in Fig. 5, row B. (c) A single subunit carved out of the 3D reference shown in panel f. (d) A 3D mask that is made from the 2D image in panel a simply by extending the density along the axial direction. (e) A 3D map that is made by cylindrically symmetrizing the density shown in panel b. (f) The 3D reference model made by multiplying the mask in panel d by the map in panel e. This reference was used to generate reference projections for a multireference alignment.
FIG. 4.
FIG. 4.
Electron micrograph of a frozen-hydrated preparation of hook-basal bodies extracted from a CW-locked mutant of S. enterica serovar Typhimurium. The arrows point to the hook-associated protein caps of three hook-basal body complexes. The L, P, M, and C rings are marked.
FIG. 5.
FIG. 5.
Class averages and variances of the C ring. Images of the hook-basal body complexes were sorted into classes based on the symmetry of the C ring (see Materials and Methods). The members of each symmetry class were averaged (left column) and the variances calculated (right column). Note how low the variance is for the sorted C rings compared to the high variance seen when the C rings (rows A and B) are averaged without sorting. The C rings are aligned on the rightmost feature (arrowhead at right top). The line along the left side accentuates the decrease of diameter with symmetry, as expected from the results of Young et al. (51) Row A shows the average of all the averaged images when aligned on the right-side feature. The left-side feature, as expected, is blurred. Note the variance in row A is low on the right and high on the left due to the misalignment of left-side features. Row B is an average over all images independent of symmetry. The alignment in this case was done using whole C rings. The variance is high because of the variation in diameter present when the averaged C rings have not been sorted according to symmetry.
FIG. 6.
FIG. 6.
Surface views of the 3D maps of the C rings from the 33-, 34-, and 35-fold symmetry classes. (Left) Side view of the C rings. (Middle) Views looking down from the top. (Right) Views looking up from the bottom. Note that the all parts of the C ring have a strong periodicity except for the innermost ring (arrows in center row).
FIG. 7.
FIG. 7.
Surface views of the 3D maps of the M rings from the 24-, 25-, and 26-fold symmetry classes. The arrows point to an inner feature that might be a part of the export apparatus.
FIG. 8.
FIG. 8.
Plot of the angular positions of the peaks detected in the 3D map of the symmetry class having 35-fold C rings and 25-fold M rings. In the reconstruction only the common fivefold symmetry was enforced. Rotational autocorrelation was used to determine the symmetry (see Materials and Methods). Shown are the positions of peaks in the autocorrelation map of the M ring (M), C ring (C), outer ring of the C ring (O), and the inner ring of the C ring (I). The lowercase m or c refers to whether the reference used in alignment was the M-ring or C-ring reference. Independent of which reference was used, there are 35 peaks for the C ring except for its inner ring, which has 25 peaks as does the M ring.
FIG. 9.
FIG. 9.
(a to d) Maps of the M and C rings made from the class of images having 25-fold M rings and 34-fold C rings. In panels a and c, the 34-fold C-ring symmetry is enforced on both the M and C rings. Note that the innermost lobe of the C ring (arrow) is weaker and relatively featureless. Note also that the M ring appears smaller because the misaveraged density is weaker. In panels b and d, the M-ring symmetry is enforced on both the M and C rings. Note that the features of the innermost ring of the C ring are strengthened and rest of the C ring features are lost. (e to g) Views from the bottom of the 3D map made from images having 33-fold C rings and 25-fold M rings, 34-fold C rings and 25-fold M rings, and 35-fold C rings and 25-fold M rings, respectively. (h to j) Side views of the maps in panels e to g. The inner ring of the C ring has had M-ring symmetry enforced. Note that there is an increasing upward tilt of the features in the inner ring of the C ring (arrows) as the C ring increases in diameter while the diameter of M ring remains unchanged.
FIG. 10.
FIG. 10.
A stereo pair showing a possible docking of the two domains of FliG into the map from the class having 34-fold C rings and 25-fold M rings. The fit was performed by eye using the graphics program O (16). The Protein Data Bank accession number for this fragment of FliG is 1LKV (5). The motor domain of FliG is docked into the inner ring feature of the C ring, and the middle domain is docked into the M ring. One FliG subunit is shown in green and the rest in red.
FIG. 11.
FIG. 11.
Two views of the rotor with a model for the stator. The part of the rotor that we attribute to FliF is shown in yellow, the part we attribute to FliG is in red, and the part of the C ring that we hypothesize to contain FliM and FliN is in blue. The stator, which contains MotA and MotB, is in pink and is based on the model of Braun et al. (4). Only eight stators are shown, although more can be fit around the periphery of the rotor.
See this image and copyright information in PMC

Comment in

  • Fine structure of a fine machine.
    Blair DF. Blair DF. J Bacteriol. 2006 Oct;188(20):7033-5. doi: 10.1128/JB.01016-06. J Bacteriol. 2006. PMID: 17015641 Free PMC article. No abstract available.

References

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    1. Braun, T. F., L. Q. Al-Mawsawi, S. Kojima, and D. F. Blair. 2004. Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli. Biochemistry 43:35-45. - PubMed
    1. Brown, P. N., C. P. Hill, and D. F. Blair. 2002. Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG. EMBO J. 21:3225-3234. - PMC - PubMed

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