Extended Data Fig. 1: Purification and characterization of recombinant proteins.

(a) Reversed-phase LC-MS spectra of purified TBA-2/TBB-2. See Luo et al. for the LC-MS spectra of purified MEC-12/MEC-7³⁰. (b) Representative Coomassie blue-stained SDS-PAGE, immunoblot analyses, and rhodamine fluorescence imaging of purified recombinant proteins. See Luo et al. for the Coomassie blue-stained SDS-PAGE, immunoblot analyses, and rhodamine fluorescence imaging of purified MEC-12/MEC-7³⁰. We performed Coomassie blue-stained SDS–PAGE analysis for every batch of protein that we purified. (c and d) Representative TIRF microscopy images showing the incorporation of FITC-labeled tubulin (green) into defects of the rhodamine-labeled TBA-2/TBB-2 (c) or MEC-12/MEC-7 (d) microtubule lattice (red) before and after the treatment of MBP-tagged human katanin P60. Scale bars: 5 µm. Similar results were observed in two independent experiments. (e) Representative cryo-EM images of GMPCPP-stabilized TBA-2/TBB-2 (top) and MEC-12/MEC-7 (bottom) microtubules showing no visible breakage or defects. Similar results were observed in 596 (TBA-2/TBB-2) and 1040 (MEC-12/MEC-7) micrographs from one cryo-EM data collection. Scale bars: 50 nm. (f and g) The FITC fluorescence intensity on per micrometer GMPCPP-stabilized TBA-2/TBB-2 microtubules (0 nM katanin: 1143 ± 374 (a.u.), n=88; 10 nM katanin: 3732 ± 1688 (a.u.), n=90; 50 nM katanin: 4780 ± 1934 (a.u.), n=78, mean ± SD are indicated) (f) and MEC-12/MEC-7 microtubules (0 nM katanin: 1515 ± 678 (a.u.), n=71; 10 nM katanin: 3077 ± 1232 (a.u.), n=73; 50 nM katanin: 5968 ± 2329 (a.u.), n=70, mean ± SD are indicated) (g) treated with different concentrations of MBP-tagged human katanin P60. The mean and SD are based on analyzed microtubules pooled from two independent experiments. The P values were calculated using a two-tailed unpaired Student’s t-test.