2.2. Sample preparation for cryo-EM

HsCKK decorated MTs were prepared as described previously (Atherton et al., 2019). Briefly, tsA201 cell tubulin was polymerised in BRB80 (80 mM PIPES, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT, pH 6.8) with 1 mM GTP at 37 °C then stabilised with 1 mM paclitaxel. At room temperature, stabilised MTs were then diluted 1/10 in BRB20 (20 mM PIPES, 2 mM MgCl2, 1 mM EGTA, 1 mM DTT, pH 6.8), adhered to holey carbon EM grids (C-Flat, Protochips Inc.) pre-glow discharged in air, and washed twice in 1 mg/mL HsCKK in BRB20. The EM grids were then blotted for 3.5 s and vitrified using a Vitrobot set at ambient temperature and 80% humidity (FEI/Thermo Fisher Scientific).

MKLP2 decorated MTs were prepared as described previously (Atherton et al., 2017b). Briefly, bovine tubulin (Cytoskeleton Inc.) was polymerised in MES polymerisation buffer (100 mM MES, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, pH 6.5) with 5 mM GTP at 37 °C then stabilised with 1 mM paclitaxel. At room temperature, stabilised MTs were adhered to holey carbon EM grids (C-Flat, Protochips Inc.), pre-glow discharged in air, and washed once in 60 μM MKLP2-MD pre-incubated in BRB20 containing 2 mM of ADP + AlF4, before blotting for 3.5 s and vitrification in a Vitrobot at ambient temperature and 80% humidity (FEI/Thermo Fisher Scientific).

For preparation of MTs decorated with the NDC domain of DCX, 5 μM bovine tubulin (Cytoskeleton Inc.) was co-polymerised at 37 °C for 30 min with 3 μM of NDC-NDC in BRB80 buffer with 1 mM GTP. The resultant NDC-NDC MT solution was applied to glow-discharged Lacey grids (Agar) and incubated for 20 s at room temperature. Then the grids were briefly blotted and 50 μM NDC-NDC solution in BRB80 buffer was applied to boost MT decoration. The grids were then transferred to Vitrobot (FEI/Thermo Fisher Scientific) and incubated there for 1 min at 30 °C and 95% humidity, before blotting for 3.5 s and plunge freezing in liquid ethane.