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. 2001 Jul;183(13):3825-32.
doi: 10.1128/JB.183.13.3825-3832.2001.

Analysis of the PilQ secretin from Neisseria meningitidis by transmission electron microscopy reveals a dodecameric quaternary structure

R F Collins  1 , L Davidsen, J P Derrick, R C Ford, T Tønjum
Affiliations

Analysis of the PilQ secretin from Neisseria meningitidis by transmission electron microscopy reveals a dodecameric quaternary structure

R F Collins et al. J Bacteriol. 2001 Jul.

Abstract

PilQ is a member of the secretin family of outer membrane proteins and is specifically involved in secretion of type IV pili in Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The quaternary structure of PilQ from N. meningitidis was analyzed by transmission electron microscopy by using a negative stain. Single particle averaging was carried out with a total data set of 650 individual particles, which produced a projection map generated from 296 particles at an estimated resolution of 2.6 nm. Oligomeric PilQ adopts a donut-like structure with an external ring that is 16.5 nm in diameter surrounding a central cavity that is 6.5 nm in diameter. Self-rotation and power spectrum analysis demonstrated the presence of 12-fold rotational symmetry, showing that PilQ is organized as a ring of 12 identical subunits. A model of the type IV meningococcal pilus fiber, based on the X-ray crystal structure of the N. gonorrhoeae pilin subunit, fitted neatly into the cavity, demonstrating how PilQ could serve as a channel for the growing pilus fiber.

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Figures

FIG. 1
FIG. 1
Detection of purified PilQ by SDS-PAGE and immunoblotting. Lane 1 shows the results of SDS-PAGE and Coomassie blue staining of purified PilQ; the purified PilQ complex is located at the well-stacking gel interface. For lane 2, after SDS-PAGE and electroblotting the filter was reacted with anti-meningococcal PilQ rabbit antiserum; the reactive material immediately below the number in this lane was the PilQ complex that was retained in the well and in the upper part of the stacking gel. The locations of the PilQ complex and monomer (∼80 kDa) are indicated.
FIG. 2
FIG. 2
(A) Electron micrograph of negatively stained, detergent-solubilized PilQ complexes (arrows). Scale bar = 20 nm. (B) Montage of 100 selected PilQ particles after Fourier filtering and contrast normalization. The montage is representative of the total sample population (n = 650).
FIG. 3
FIG. 3
(A) Nonsymmetry averaged projection of PilQ (n= 296; ascendant classification threshold, 0.04; sampling interval, 0.47 nm). Scale bar = 6 nm. (B) Stain density across the diameter of the averaged PilQ projection.
FIG. 4
FIG. 4
Multiple-peak orientation search for the PilQ average (see Fig. 3A). A full ring search detecting 360 maximum peaks was employed. For relative peak intensities of >0.04, the variation from exact 30° repeats was no greater than ± 5°.
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