Radial mass analysis of the flagellar filament of Salmonella: implications for the subunit folding

X-ray fiber diffraction patterns of the R-type straight flagellar filament of Salmonella typhimurium SJW1655 strain showed layer-lines with an axial spacing of 1/437 A-1, which could be resolved only due to very small disorientation angles (< 2 degrees) of the filaments in oriented sol specimens. Although the equatorial layer-line was situated between the relatively strong first layer-lines right above and below it, these small disorientation angles and a new method of two-dimensional angular deconvolution allowed us to determine the equatorial layer-line intensities quite accurately. The equatorial data were phased by using the amplitude difference between the native flagellar filament and its heavy atom derivatives. One of the heavy-atom derivatives was prepared by introducing a cysteine residue by site-directed mutagenesis and applying a mercury compound. From the equatorial structure factors, the radial density distribution of the filament was calculated at 11 A resolution. A prominent feature was two pairs of high density peaks at radii of around 25 and 45 A and a deep density trough between them, which corresponds to the concentric double tubular structure in the core region that has been found in the density map recently deduced by helical image reconstruction from electron micrographs of frozen hydrated filaments. The molecular masses were estimated for four radial segments that correspond to the morphological domains identified in the map of helical image reconstruction. Then the domains were assigned to sequence positions by correlating the estimated masses with those of proteolytic fragments of flagellin. The assignment is consistent with the distributions of secondary structures and in particular alpha-helical coiled-coils that were predicted from the sequence. It also helps to understand how the polymerization behaviour is affected by truncation of the disordered terminal regions of flagellin and why mutations in a specific region are responsible for changes in the polymorphic shape of the filament.