Principle of defined divalent streptavidins. Numbering of subunits in the streptavidin tetramer, with each monomer colored separately and biotin in space-fill (from PDB ID: 3RY2). Below are arrangements of 1,2 or 1,3 biotin-binding sites (biotin-bound monomers, green; non-binding monomers, blue), showing relative orientations and distances between the biotin carboxyl group carbons.