Structure of bacterial flagellar filaments at 11 A resolution: packing of the alpha-helices.

Recent advances in the analysis of electron micrographs of frozen, hydrated bacterial filaments have allowed us to average data from more than 150 images and to reconstruct the bacterial flagellar filament of Salmonella typhimurium at a resolution of approximately 11 A. In addition to the outermost features seen in earlier lower resolution maps of the filament, we find a pair of concentric tubes which surround a approximately A diameter channel at the center of the structure. The walls of these tubes are composed of rod-like features which we have interpreted as columns of individual alpha-helices stacked end-to-end. Each column runs approximately parallel to the helix axis. The wall of the innermost tube, at a radius of approximately 20 A, is formed from 11 such columns. The wall of the second tube is formed from 22 columns which occur alternately at radii of approximately 43 and approximately 47 A. The two concentric tubes are held apart by spacers. These are short, rod-like features, which run approximately parallel to the helix axis. We have interpreted these as additional alpha-helices. By symmetry, each flagellin monomer contributes an alpha-helix to the inner tube, two alpha-helices to the outer tube and a fourth alpha-helix to the spacer. We have tentatively assigned one type of alpha-helix in the outer tube to the approximately 30 C-terminal residues of flagellin while the remaining three alpha-helices are assigned to the approximately 70 N-terminal residues. This interpretation of the reconstruction is consistent with available biochemical, biophysical and amino acid sequence information. We also present details of improved methodology to extract and evaluate the original data and also to assess the statistical significance of features in the three-dimensional map.

Fibrinogen structure in projection at 18 A resolution. Electron density by co-ordinated cryo-electron microscopy and X-ray crystallography.

Electron microscope images of frozen-hydrated crystals of a proteolytically modified fibrinogen show excellent preservation of the structure. An electron density map of the key centric projection of the crystal at 18 A resolution has been obtained by combining the phases derived from cryo-electron microscopy with X-ray amplitudes. Simulation methods developed in earlier studies have been used to interpret the map. In contrast to the earlier images, the map allows us to visualize the coiled-coil region of the molecule and possible substructure in the beta domains. The map also shows that there is a marked difference in density in the two regions corresponding to the molecular ends where the gamma domains interact. A possible interpretation of this finding is provided by assuming substructure in the gamma domains and the breaking of molecular symmetry where these domains interact. Some additional constraints useful for the determination of the three-dimensional structure were obtained from cryo-electron micrographs of a perpendicular view at 25 A resolution. Implications of this working model for the molecular length and contacts in the filaments in both the crystal and fibrin are described. The data used here will be valuable as a starting point for obtaining the three-dimensional structure.