His6-Z-TEV-LRRK2 was expressed and purified as described in a previous protocol

LRRK1 expression and purification

Prepare LRRK2 buffer. Keep it at 4ºC.

20 millimolar (mM) HEPES pH=7.4

150 millimolar (mM) NaCl

2.5 millimolar (mM) MgCl2

20 micromolar (µM) GDP

0.5 millimolar (mM) TCEP

Spin down purified LRRK2 (10000 rcf, 4°C, 10 minutes). Leave protein on ice afterward.

For the best result, keep protein on ice and reduce the amount of time between spinning

and freezing cryo-EM samples.

Thaw E11 DARPin and spin it down. Measure its concentration.

Dilute inhibitors (diluted in 100% DMSO) to an intermediate desired concentration using LRRK2 buffer.

Based on the initial and final LRRK2 concentration, calculate the necessary volume of E11 DARPin to get a proportional ratio LRRK2:E11 DARPin 1:1.25 in the sample. After that, add either MLi-2 or GZD-824. Dilute to your final desired LRRK2 concentration using LRRK2 buffer (150 mM NaCl).

Incubate 10 minutes at RT. Afterward, keep it on ice until grid preparation.

We used UltraAuFoil Holey Gold 2/2 200 mesh grids and plasma cleaning them in the Solarus II (Gatan) using the QuantiFoil Au preset.

Apply 3 to 3.5 microliters (µl) of sample and plunge freeze. We used a

Vitrobot (FEI) to blot away excess sample and plunge freeze in ethane liquid. (In

our case, we use 4 seconds as a time blot as 20 sec as a wait time and 4 as a blot force, but these

parameters are slightly different from one Vitrobot to another. I would try

with the Vitrobot parameters already tested in your machine first).

Store grids in liquid nitrogen until ready for imaging