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 <i>de novo</i> 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 (<i>S</i> = 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, <i>N</i>-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.