Preparation of LRRK2 RCKW cryo-EM grids
Mariusz Matyszewski
Abstract
This is Leschziner's Lab updated protocol for making cryo-EM grids for LRRK2 RCKW. This protocol, when using lower protein concentration, results in better monomer and dimer formation than the old protocol.
Before start
Decide which protein concentration to use, and create the proper LRRK2 buffers in order to obtain the right salt concentration (80 mM NaCl).
Steps
Preparing Sample
Spin down purified LRRK2 RCKW. 10000rcf,4°C
Leave protein on ice afterwards.
Freezing Grids
Plasma clean grids.
We used UltrAuFoil Holey Gold 1.2/1.3 300 mesh grids and plasma cleaned them in the Solarus II (Gatan) using the QuantiFoil Au preset.
Dilute samples to desired concentration in the LRRK2 buffer . Make sure final salt is at 80 mM NaCl.
For best results, make 10µL samples, good for freezing 2 grids. This is to minimize time spent outside of storage buffer, reducing aggregation.
(Optional) If using inhibitors, let them incubate on ice in the final LRRK2 buffer before plunge freezing.
Apply protein to grids and plunge freeze.
We used a Vitrobot (FEI) to blot away excess sample and plunge freeze
Store grids in liquid nitrogen until ready for imaging.
