MyoA:ELC:MTIP and Act1 were expressed and purified as previously described [10 (link),24 (link)]. For Act1 samples, ammonium acetate was removed by a spin column, and Act1 (13.1 μM) was polymerized in the presence of 13 μM jasplakinolide by adding 10X KMEI, in final 1x concentration of 10 mM Hepes, pH 7.5, 50 mM KCl, 4 mM MgCl2, 1 mM EGTA. Polymerized filamentous Act1 was diluted down to 0.13 μM, and treated with apyrase (77 μg/ml) for 20 min before addition of the MyoA:ELC:MTIP complex in a 1:1 ratio.
For Act1-only samples, no apyrase was used. After 30 min incubation, negatively stained or cryo-EM grids were prepared. The quality of the decorated filaments was first checked with negative staining, and after confirming the presence of decorated Act1 filaments, frozen-hydrated samples were prepared on Quantifoil 2/2, 200 mesh in-air glow-discharged grids using an Mk III Vitrobot (FEI). 3 μl of sample was applied on a grid and blotted for 3 s before plunge-freezing in liquid ethane.
For Act1-only samples, no apyrase was used. After 30 min incubation, negatively stained or cryo-EM grids were prepared. The quality of the decorated filaments was first checked with negative staining, and after confirming the presence of decorated Act1 filaments, frozen-hydrated samples were prepared on Quantifoil 2/2, 200 mesh in-air glow-discharged grids using an Mk III Vitrobot (FEI). 3 μl of sample was applied on a grid and blotted for 3 s before plunge-freezing in liquid ethane.
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