Microtubules are subject to several post-translational modifications, such as tyrosination. Tubulin Tyr ligase (TTL) catalyses the re-addition of a Tyr residue to the carboxyl terminus of detyrosinated α-tubulin, and this has several important cellular roles. Prota et al. identified various structures of TTL in complex with tubulin by X-ray crystallography. They found that TTL specifically recognizes the curved conformation of unassembled α-tubulin–β-tubulin dimers and binds tubulin adjacent to the dimer interface. The proximity of α-tubulin to the TTL active site and the observation that α-tubulin contains two C-terminal acidic residues necessary for TTL binding explain how TTL discriminates between α- and β-subunits. These structures also show why TTL cannot bind straight microtubules and thus selectively modifies unassembled tubulin. Notably, the tubulin-contacting residues are conserved among TTL orthologues. Moreover, nucleotide binding was important for the shaping of the TTL active site, suggesting that tyrosination reactions are coordinated with nucleotide exchange.