Classification of VHHs by CDR3 loop structures. (A) The structure of VHHs is classified into three types based on the conformation of CDR3; Upright, Half-Roll and Roll (PDB ID: 5j1s, 6ru3, and 2p45). (B,C) The distances between Cα atoms of CDR3 loop and FR2 (distance between H46 residue and (n − 5)th residue with H102 as (n)th residue) and pseudo-dihedral angles of CDR3 C-terminal (dihedral angle consisting of the four Cα atoms of the (n + 1)th, (n)th, (n − 1)th, and (n − 2)th residues, with H102 as the (n)th residue) were calculated from structures and the value was plotted. (B) The structures of CDR3 were classified into the three groups according to type (Upright, Roll, and Half-roll) based on the distance and pseudo-dihedral angle. The VHHs belonging in the Upright group (pink dots) had a distance of 15Å or more and an angle of 140° to 360°, the VHHs in the Roll group (green dots) had a distance of 10Å or more and an angle of less than 140°, and the other VHHs were classified as the Half-Roll type (blue dots). (C) The CDR3 of human VH (gray dots) from a conventional VH–VL complex and VHH (yellow dots) are shown.