Electron microscopy (EM) analysis of LRRK2-Nanotube assembles

Prepare samples in a PCR tube with 300nanomolar (nM) LRKK2, 20micromolar (µM) lipid nanotubes and 1millimolar (mM) GMPPNP.

Incubate samples at 37°C for 0h 30m 0s.

Glow-discharge carbon-coated grids (25 mA, 0h 0m 45s) during the sample incubation time.

Place the discharged grids on a piece of parafilm.

After incubation, apply 6µL of the samples to the grid and adsorbed on the grid for 0h 5m 0s at Room temperature.

Blot the grid with filter paper and stained with 2% uranyl acetate for 0h 0m 40s.

Dry the grid with filter paper.

Collect images using a Talos L 120C TEM microscope at 80 kV with Velox software and a 4k × 4K Ceta CMOS camera (Thermo Fisher Scientific).

Dialyze freshly purified LRRK2 into the Low salt buffer.

After dialysis, incubate LRRK2 (2micromolar (µM)) with the kinase inhibitor MLi-2 (5micromolar (µM)) for 0h 10m 0s On ice.

Add 20micromolar (µM) lipid nanotubes into the mixture above and further incubate for 1h 0m 0s at Room temperature in the presence of 1millimolar (mM) GTP.

Glow-discharge C-flat™ holey carbon gold grids (CF-1.2/1.3-3Au) (15mA, 0h 0m 45s) during the sample incubation time, then place the discharged grids on a piece of parafilm.

After incubation, apply 4µL of the samples to the grid.

Plunge-freeze sample-loaded grids in liquid ethane-propane mixture using a Vitrobot Mark IV (FEI) with the following parameters: blot force, 0; blot time, 0h 0m 1s; wait time, 0h 0m 30s; drain time, ; humidity, 100%.

Collect cryo-EM micrographs on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV, equipped with a post column GIF quantum energy filter and a Gatan K3 Summit DED camera (Gatan, Pleasanton, CA, USA).

Perform the data collection with the SerialEM software. Record movies in super-resolution mode with a physical pixel size of 1.098 A˚ (super-resolution pixel size is 0.549 A˚) and a defocus range of -1 µm to - 3 µm .