Glutamylation, the most prevalent tubulin posttranslational modification, marks 
stable microtubules and regulates recruitment and activity of microtubule- 
interacting proteins. Nine enzymes of the tubulin tyrosine ligase-like (TTLL) 
family catalyze glutamylation. TTLL7, the most abundant neuronal glutamylase, 
adds glutamates preferentially to the β-tubulin tail. Coupled with ensemble and 
single-molecule biochemistry, our hybrid X-ray and cryo-electron microscopy 
structure of TTLL7 bound to the microtubule delineates a tripartite microtubule 
recognition strategy. The enzyme uses its core to engage the disordered anionic 
tails of α- and β-tubulin, and a flexible cationic domain to bind the 
microtubule and position itself for β-tail modification. Furthermore, we 
demonstrate that all single-chain TTLLs with known glutamylase activity utilize 
a cationic microtubule-binding domain analogous to that of TTLL7. Therefore, our 
work reveals the combined use of folded and intrinsically disordered substrate 
recognition elements as the molecular basis for specificity among the enzymes 
primarily responsible for chemically diversifying cellular microtubules.