Extended Data Fig. 7 ∣. Distance between MTBH2 and β-tubulin tails on the microtubule and mass spectra showing subtilisin and subtilisin mediated proteolytic removal of β-tubulin tails from unmodified human microtubules.

a. Schematic showing the distance between the start of the β-tubulin tail (yellow star) and the MTBH2 (cyan star). The disposition of the β-tubulin tail (its length and position as it emerges from the tubulin body) is such that the MTBH2 is able to interact only with the β-tail from the lateral tubulin dimer because the most C-terminal glutamate that cross-links to the MTBH2 lysine cannot reach closer than ~30 Å from the C-terminus of MTBH2, even when we assume that the β-tail adopts a completely extended random coil conformation (3.8 Å Cα to Cα distance) which would give it the greatest span (the distance between the start of the β-tail and the MTBH2 on the same tubulin dimer is ~75 Å). Even the most C-terminal glutamate residue in the β-tail of the same dimer on which TTLL6 sits cannot reach closer than ~18 Å from MTBH2. In contrast, the Cα of the terminal Lys residue of TTLL6 MTBH2 is positioned ~9 Å away from the last β-tubulin residue visible in our structure (D427, and the start of the flexible β-tubulin tail) that belongs to the laterally adjacent β-tubulin. b,c. LC-MS spectra of wild-type (b) and subtilisin (c) treated unmodified human microtubules used in Fig. 4 (Methods). The tubulin isotypes and the different β-tubulin species after subtilisin treatment are indicated.