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Colonization factor CFA/I defines the major adhesive fimbriae of enterotoxigenic Escherichia coli and mediates bacterial attachment to host intestinal epithelial cells. The CFA/I fimbria consists of a tip-localized minor adhesive subunit, CfaE, and thousands of copies of the major subunit CfaB polymerized into an ordered helical rod. Biosynthesis of CFA/I fimbriae requires the assistance of the periplasmic chaperone CfaA and outer membrane usher CfaC. Although the CfaE subunit is proposed to initiate the assembly of CFA/I fimbriae, how it performs this function remains elusive. Here, we report the establishment of an in vitro assay for CFA/I fimbria assembly and show that stabilized CfaA-CfaB and CfaA-CfaE binary complexes together with CfaC are sufficient to drive fimbria formation. The presence of both CfaA-CfaE and CfaC accelerates fimbria formation, while the absence of either component leads to linearized CfaB polymers in vitro. We further report the crystal structure of the stabilized CfaA-CfaE complex, revealing features unique for biogenesis of Class 5 fimbriae. Author summary Colonization factor antigen I (CFA/I) is a representative member of alternative chaperone-usher fimbriae in enterotoxigenic Escherichia coli, a major cause of travelers' diarrhea. During assembly, the tip-adhesive subunit CfaE might conceivably serve as an initiator, but the mechanism is not well understood. We demonstrate that the chaperone-tip adhesin complex CfaA-CfaE is essential in early fimbriae growth. The crystal structure of this complex reveals a specific chaperone-anchoring motif and a functional inter-domain loop in CfaE. Our findings suggest that the interaction of the CfaA-CfaE complex with CfaC is responsible for control of the usher plug domain movement and facilitates the binding of CfaE adhesin domain into the CfaC lumen. Collectively, our data demonstrate the crucial role of CfaA-CfaE in CFA/I fimbriae production, providing guidance for further development of novel agents targeting CFA/I fimbriae and against ETEC infection.