STRUCTURAL BASE OF ASSEMBLY AND POLYMORPHISM OF BACTERIAL FLAGELLAR FILAMENT. Keiichi Namba, Yuko Mimori, Ichiro Yamashita, Ferenc Vonderviszt, International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd., 3-4 Hikaridai, Seika 619-02 Japan

A complementary use of X-ray fiber diffraction and electron cryomicroscopy has allowed us to deduce the domain structure of flagellin subunit in the flagellar filament, which reveals overall folding of flagellin and direct interaction of the termini in the very inner core of the filament. Flagellar filaments are formed by self-assembly process and are known to be polymorphic, being able to take various supercoiled forms as well as the two distinct straight forms. To understand the mechanisms of self-assembly and polymorphism, structure analysis toward atomic resolution is underway. Electron cryomicroscopy and helical image reconstruction were used to analyze the structures of various straight filaments at around 10 Å resolution. The layer-line spacings and symmetries of the filaments used in the EM analyses were obtained from X-ray fiber diffraction patterns of well oriented sols with disorientation angles less than 1 degree, which were prepared by liquid crystallization and magnetic orientation of flagellar sols. By carefully comparing the filaments of intact flagellins with those reconstituted with various flagellins of terminal truncations and central deletions, structural domains were assigned to sequence positions. In particular, a direct terminal interaction was found essential for the correct folding of large terminal regions that form the very inner core of the filament, which is in turn essential for the polymorphic ability.

A two-dimensional extension of the angular deconvolution method was applied to process X-ray diffraction patterns from well-oriented sols, which allowed us to extract layer-line amplitude distribution reliably. The phase data from the EM analyses were combined with X-ray amplitudes from the native and heavy atom derivatives of the filament to locate heavy atom binding positions in difference Fourier maps. The multiple isomorphous replacement phasing procedure is being applied to deduce electron density maps at higher resolution available in the X-ray data.