Updated project metadata. Many bacteria express filaments called type IV pili on their surface, which are involved in motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Type IV pili are comprised of a helical assembly of repeating pilin subunits that allow both flexibility and strength. They are therefore powerful structures that enable bacterial proliferation and genetic adaptation, potentially leading to the development of pathogenicity and antibiotic resistance. They are also targets for drug development. By electron cryo-microscopy and mass spectrometry, we show that the bacterium Thermus thermophilus produces two forms of type IV pilus, differing in structure and protein composition. We have determined the structures of both and built atomic models, which reveal a new pilin, how the subunits assemble and their glycosylation patterns. We also delineate the roles of the two filaments in promoting twitching and natural transformation.