Cryo-EM sample preparation for full length LRRK2(I2020T):MLi-2/GZD-824-E11 DARPin complex
Marta Sanz Murillo, Amalia Villagran Suarez
Abstract
Guide for cryo-EM grid preparation of FL-LRRK2 bound to Type-I/Type-II inhibitors
Before start
Before starting cryo-EM grid preparation section: Set up Vitrobot at 95% humidity and 4C. Put new paper blot
Steps
Protein purification
His6-Z-TEV-LRRK2 was expressed and purified as described in a previous protocol
Sample preparation
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.
cryo-EM 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
