biostudies-literatureDang VTMichigan Technology Tri-CorridorNational Institute of General Medical SciencesNIGMS NIH HHS2304-2308https://www.ebi.ac.uk/biostudies/studies/S-EPMC12857771biostudies-literatureUnknown62(7)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.Chemical communications (Cambridge, England)β-barrels from short macrocyclic peptides.PMC12857771R35GM154793085P1000817R35 GM154793McElheny DDang VTNguyen AIMartynowycz MWfalseβ-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.2026-01-01T00:00:00Z2026 Jan2026-06-16T07:26:39.788Z2026-06-16T03:10:09.175ZS-EPMC128577714148962610.1039/d5cc06640a