Extended Data Fig. 1 ∣. Cryo-EM image processing.

a. Initial image processing was performed in RELION⁸³. In total, 2,771 movies were motion corrected and summed⁹⁸; high-quality micrographs were selected based on CTF estimation⁸⁰ and visual inspection for manual picking of microtubule segments. b. Representative cryo-EM micrograph from one of 2,771 movies of microtubules decorated with TTLL6. Decoration is highly heterogeneous. Scale bar, 25 nm. c. Initial reconstruction of microtubule decorated with TTLL6 using MiRP⁸¹. Microtubule segments were sorted based on protofilament number; most common rotation angle, x/y shifts and seam positions were assigned to each microtubule segment. d. C1 reconstruction of the TTLL6 bound microtubule. TTLL6, gold, binds every 80 Å, highlighted by the double arrow, along the microtubule lattice, blue. e. Protofilament refinement⁵⁴ improved map quality for the microtubule and MTBH1-2 in TTLL6. For each microtubule segment all but the signal from two adjacent protofilaments was substracted to generate a two protofilament stack, followed by refinement of particle coordinates. f. Local resolution estimates show the map reaching 3 Å resolution in the microtubule and MTBH1-2 regions and poorly resolved density for the rest of TTLL6. MTBH1-2 is highlighted by a dashed rectangle. Local resolution was determined via Monores⁸² followed by LocalDeblur⁹⁹. g. Two protofilament, 2PF, refinement protocol improved definition of the C-terminus of MTBH2 in comparison to a protocol where each particle contained signal for just one protofilament, 1PF. h. Focused classification of TTLL6 improved its definition. Refinement of the minor class yielded a TTLL6 map with local resolution estimates ranging from ~5 Å to 14 Å. i Fourier shell correlation (FSC) curves for each microtubule reconstruction depict their nominal resolution. FSC estimates were calculated at 0.143 criterion. Resolution for the microtubule lattice improved from 3.8 Å, MiRP protocol, to 3.6 Å, two protofilament refinement protocol.