High-resolution structures of macromolecular complexes offer unparalleled insight into the workings of biological systems and hence the interplay of these systems in health and disease. We have adopted a multifaceted approach to understanding the pathogenically important structure of P-pili, the class I adhesion pili from pyelonephritic Escherichia coli. Our approach combines electron cryomicroscopy, site-directed mutagenesis, homology modeling, and energy calculations, resulting in a high-resolution model of PapA, the major structural element of these pili. Fitting of the modeled PapA subunit into the electron cryomicroscopy data provides a detailed view of these pilins within the supramolecular architecture of the pilus filament. A structural hinge in the Nterminal region of the subunit is located at the site of a newly resolved electron density that protrudes from the P-pilus surface. The structural flexibility provided by this hinge is necessary for assembly of P-pili, illustrating one solution to construction of large macromolecular complexes from small repeating units. These data support our hypothesis that domain-swapped pilin subunits transit the outer cell membrane vertically and rotate about the hinge for final positioning into the pilus filament. Our data confirm and supply a structural basis for much previous genetic, biochemical, and structural data. This model of the P-pilus filament provides an insight into the mechanism of assembly of a macromolecular complex essential for initiation of kidney infection by these bacteria.electron cryomicroscopy ͉ fimbriae ͉ macromolecular assembly ͉ domain swapping ͉ helical reconstruction B acterial adhesion is a key initiating step in the infection process, which is frequently correlated with the ability of pathogenic bacteria to colonize host tissue (1, 2). P-pili are necessary and sufficient for binding of pyelonephritic Escherichia coli to their human host (3), so detailed insight into the assembly process and 3D structure of these pili provides valuable information for the design of therapeutics that prevent or eliminate infection by interfering with this binding.Our understanding of cellular solutions to mechanical problems is aided greatly by investigating macromolecular complexes on the scale of P-pili. Here we elucidate how a bacterium builds an extracellular structure larger than the channel through which it must pass. Transport of the proteins of uropathogenic pilins occurs via the general secretory pathway, by which pilins cross the inner cell membrane and are then chaperoned across the periplasm. At the outer membrane the pilins are translocated through a pore-forming usher and assembled into the pilus by proximal addition to the growing helical filament. The pore is large enough to accommodate a single pilin subunit in its native conformation but cannot accommodate the assembled helical filament ( Fig. 1; for a review of pilus assembly, see ref. 4).Class 1 pili, including P-pili, type 1 pili, and Hib pili, are all helical structures 7-8 nm in diame...

SummaryTo survive the harsh environment of a churning intestinal tract, bacteria attach to the host epithelium via thin fibers called pili (or fimbriae). Enterotoxigenic Escherichia coli expressing CFA/I pili and related pili are the most common known bacterial cause of diarrheal disease, including traveler's diarrhea. CFA/I pili, assembled via the alternate chaperone pathway, are essential for binding and colonization of the small bowel by these pathogenic bacteria. We elucidate unique structural features of CFA/I pili that appear to optimize their function as bacterial tethers in the intestinal tract. Using transmission electron microscopy of negatively stained sample in combination with iterative threedimensional helical reconstruction methods for image processing, we have determined the structure of the CFA/I pilus filament. Our results indicate that strong end-to-end protein interactions and weak interactions between the coils of a sturdy spring-like helix provide the combination of strength, stability, and flexibility required to sustain bacterial adhesion and incite intestinal disease. We propose that CFA/I pili behave like a spring to survive the harsh environment of a churning intestinal tract, thereby persisting long enough for these bacteria to colonize the host epithelium and cause enteric disease. KeywordsAdhesion Pili; Fimbriae; ETEC; Helical Reconstruction; Electron Microscopy and Image Processing Enteric bacterial infection is a significant worldwide health risk, particularly for infants and small children. Enterotoxigenic Escherichia coli (ETEC) are the most frequently isolated cause of community-acquired childhood diarrhea in the developing world, accounting for over 200 million episodes of illness 1; 2 and 380,000 deaths 2 per year, and the most common cause of travelers' diarrhea 3 . Bacterial adhesion is a key initiating step in the infection process, frequently correlated with the ability of bacteria to colonize host tissue. Adhesion is mediated by colonization factor antigens, with Colonization Factor Antigen I (CFA/I) a commonly isolated virulence factor (see, e.g. review by Qadri et al. 4 , and earlier references 5; 6 ). The long, thin surface filaments of CFA/I pili (also called "fimbriae") mediate attachment of ETEC to the small intestine during the earliest pathogenic stages in the host, and are essential for binding and colonization of the small bowel 7 , yet few details of their fine structure are known.* To whom correspondence should be addressed. E-mail: bullitt@bu.edu The views expressed in this article are those of the authors and do not necessarily reflect the official position of the National Institutes of Health, the Department of the Navy, Department of Defense, nor the U.S. Government.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it i...

Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 Å in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 Å along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.

An explicit equation for X-ray diffraction by a ®nite one-dimensional paracrystal is derived. Based on this equation, the broadenings of re¯ections due to limited size and disorder are discussed. It depicts the paracrystalline diffraction over the whole reciprocal space, including the small-angle region where it degenerates into the Guinier equation for small-angle X-ray scattering. Positions of diffraction peaks by paracrystals are not periodic. Peaks shift to lower angles compared to those predicted by the average lattice constant. The shifts increase with increasing order of re¯ections and degree of disorder. If the heights and widths of the paracrystalline diffraction are treated as reduced quantities, they are functions of a single variable, N 1a2 g. The width of the ®rst diffraction depends mostly on size broadening for a natural paracrystal, where N 1a2 g 0.1±0.2. A method to measure the paracrystalline disorder and size using a single diffraction pro®le is proposed based on the equation of paracrystal diffraction. An initial value of size may be obtained using the Scherrer equation, that of the degree of disorder is then estimated by the à law. Final values of the parameters are determined through least-squares re®nement against observed pro®les. An equation of diffraction by a polydisperse one-dimensional paracrystal system is presented for`box' distribution of sizes. The width of the diffraction decreases with increasing breadth of the size distribution.

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