Structure and Assembly of a Trans-Periplasmic Channel for Type IV Pili in Neisseria meningitidis
Figure 5
Determination of PilQ oligomer structure by single particle averaging: Raw data image and class averages.
A. Frozen-hydrated specimen of PilQ complexes resuspended in a dodecyl maltoside-containing buffer at 1 mg/ml. The particles in the 1300 nm diameter hole in the carbon support film were selected interactively for image processing and single particle alignment, and then a preliminary model was generated by selecting projections with bilateral symmetry as well as 12-fold rotational symmetry. This model was then used as an alignment reference to which all particles were aligned, after which a refined model was generated. After several rounds of iterative refinement, no further improvement in the model was detected (as judged by Fourier Shell Correlation between the Nth and (N-1)th iteration). B. Comparison between different orientations of the final model (even numbered projections) and averages of all the particles best corresponding to those projections (subsequent odd numbered projection averages). Deviations between the odd and subsequent even numbered projections reflect the errors in the processing procedure, especially with respect to classification and rotational and translational alignment, and are a useful separate measure of the resolution and reliability of the structural data.