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. 2003 Apr;185(8):2611-7.
doi: 10.1128/JB.185.8.2611-2617.2003.

Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy

Richard F Collins  1 , Robert C Ford, Ashraf Kitmitto, Ranveig O Olsen, Tone Tønjum, Jeremy P Derrick
Affiliations

Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy

Richard F Collins et al. J Bacteriol. 2003 Apr.

Abstract

The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.

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Figures

FIG. 1.
FIG. 1.
PilQ particles visualized under negative stain. (A) Tilt pairs of PilQ particles at 0° and 40°, negatively stained with 2% uranyl acetate and recorded under low-dose conditions. Arrows indicate particles presenting the face-on donut view. Scale bar, 30 nm. (B) Montage of particles presenting putative side-on views of PilQ. Measurements of individual particles found widths and heights in the range of 15 to 16.5 nm and 18 to 20 nm, respectively. Scale bar, 15 nm. (C) 2-D projection maps of the two major class averages (n = 200 and 303) of particles selected for volume back projection. Maps are low-pass filtered to 2 nm and contoured at 20 levels. Scale bar, 10 nm.
FIG. 2.
FIG. 2.
The distribution of accumulated negative stain around the circumference of the merged PilQ volume demonstrates 12-fold symmetry. The unsymmetrized PilQ volume (n = 503) displayed as a density map (in blue at 2σ above the mean density value) is positioned to correspond to the donut-like projections in Fig. 1. Twelve peaks of negative-stain volume (shown in red and yellow at 1.5σ and 2σ, respectively, above the mean negative-stain density) occur at approximate 30° intervals around the PilQ ring and are labeled 1 to 12. The boundary between the regions in the volume corresponding to the PilQ complex and the surrounding stain pockets is shown by the dotted line. Scale bar, 10 nm.
FIG. 3.
FIG. 3.
Surface-rendered 3-D volumes of PilQ with 12-fold rotational symmetry applied. Volumes are displayed at a threshold of 2σ above the mean volume density. Scale bar, 10 nm. (A) Face-on view corresponding to the donut-like projection in Fig. 1. (B) Rotation of the face-on view of the PilQ complex 90° through the y axis. Viewed from the side, the PilQ volume has two distinct regions: a ring (R) region connected to an underlying plug (P) region. (C) A cross-sectional side-on view of the PilQ volume with 50% of foremost volume removed through the z axis to reveal the structure of the internal cavity. The constriction of the central cavity may be seen at the bottom of the complex and is indicated by arrows. Ribbon plots of the atomic model of the N. meningitidis Tfp fiber are displayed to the right in a side-on and face-on view at the same scale as the PilQ volumes. Five subunits (one helical turn) are shown.
See this image and copyright information in PMC

References

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