β-barrels from short macrocyclic peptides

β-Barrels are ubiquitous motifs in protein structures, but the fundamental rules underlying their formation are unclear, and their de novo design remains highly challenging. Small peptides that form barrels are especially scarce. Here, we report barrels with the shortest staves (6 residues, ∼60% of previous record) and smallest shear number (S = 4) so far, formed from 12-residue macrocyclic peptides. The miniature barrel has anomalous structural features, demonstrated by solution phase and crystallographic characterization; there is a pronounced and essential backbone kink imparted by an achiral residue, N-methylglycine, as well as four structural water molecules stitching the seams of the barrel. These results provide insights into how extremely short sequences could form barrel assemblies.