Enterotoxigenic E. coli (ETEC) express CFA/I pili on their surface. These pili are ∼1 μm long helical filaments, 7.4 nm in diameter. Isolated pili either retain their helical structure and are visualized as straight filaments (black arrows) gently curved (black arrowheads), or sharply bent filaments (white arrowheads), or appear to be unwinding into extended thin fibrillar structures (white arrows). Isolated CFA/I pili were placed on carbon-coated copper grids, stained with 1% uranyl acetate, and imaged in a Philips CM12 or FEI TF20 electron microscope. Images were taken at 60,000x or 50,000x, respectively. Magnification bar, 250Ǻ.

3-D reconstruction of CFA/I pili showing that the right-handed helical filament has a repeat of 19 CfaB pilin subunits in 6 turns of the helix (3.17 subunits per turn) with a 0.83 nm rise per subunit along the helical axis. Shown here are (a) a surface view of the structure, (b) a projection view which mimics the original EM data, (c) a surface view showing only the high electron density of the structure; this view is helpful for viewing the bi-lobed domain structure of the CfaB subunit (white and black lines) and to see the progression of subunits along and around the helical filament: subunit n is adjacent to n+1, subunit n+2 is around the back of the filament, and subunit n+3 is the subunit which stacks above subunit n in the coiled helical structure. (d) a cross-section of the helix is shown as a grey-scale map; this view is taken at vertical location marked by the arrow in panel (b). Magnification bar in panel (d), 50 Ǻ, is for all panels.

(a) Sample CFA/I pili “particles”, including segments that will be discarded: row 2, column 1 due to the bend in the filament, and row 2, column 5 due to the crossing filament. The particle in row 4, column 3 will be discarded if it is not centered properly in subsequent iterative cycles. Images for the reconstruction were all taken on an FEI TF20 electron microscope, and selected using the EMAN boxer software 26, and processed using a modified version of the IHRSR 22 in combination with the SPIDER/WEB software package 23. Magnification bar, 50 Ǻ. (b) Differences in electron density of symmetry-related regions of five sample starting models. Shown here are starting models with a rotation angle of 112° between subunits, and a rise per subunit of 5.6-11.2 Ǻ in steps of 0.8 Ǻ. The two models with the lowest electron density differences in the final iteration have converged to the same solution from different starting symmetries. The full dataset was used to test the best and second-best starting models. (c) The electron density differences were consistently lower in the best model. (d) The second-best starting model did not converge to a solution, even after 100 iterative cycles, whereas the best starting model (e) converged quickly. In panels (d) and (e) both the rotation angle (lighter line) and the rise per subunit (darker line) are shown in the graphs. In all panels, the abscissa corresponds to the iterative cycle number.

To compare the structures of CFA/I pili, P-pili, and Hib pili, surface views of enterotoxigenic E. coli CFA/I pili (left, green), uropathogenic E. coli P-pili (middle, pink) and oronasopharyngeal H. influenzae Hib pili (right, blue) are shown at high electron density thresholds. These surface views show only the strongest protein density, and for clarity, the far side of each helix is not shown. The strong connections between subunits around the helical axis of CFA/I pili (left panel, yellow arrow), and the relatively weak connections between subunits that are the stacked coils of the helix (left panel, white arrow), are compared with P-pili (center panel) and Hib pili (right panel). Note that Hib pili have essentially no connections around the helical axis, and therefore the yellow arrow appears to point to a black region of the map. Under stress a morphological transition may confer limited unstacking properties to CFA/I pili, allowing motion without breaking, thereby supporting sustained binding of ETEC in the gastrointestinal tract.