Sep 09, 2022

Public workspacePreparation of LRRK2 RCKW cryo-EM grids

 Forked from Preparation of LRRK2 RCKW trimer cryo-EM grids
DOI
dx.doi.org/10.17504/protocols.io.kxygxp7ywl8j/v1
  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
Mariusz Matyszewski
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DOI: dx.doi.org/10.17504/protocols.io.kxygxp7ywl8j/v1
Protocol Citation: Mariusz Matyszewski 2022. Preparation of LRRK2 RCKW cryo-EM grids. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxp7ywl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 31, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 46832
Keywords: cryo-EM, LRRK2, structural biology, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
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.
Materials
LRRK2 Buffer:
  • Concentration20 millimolar (mM) HEPES pH 7.4
  • Concentration80 millimolar (mM) NaCl
  • Concentration0.5 millimolar (mM) TCEP
  • Concentration5 % volume Glycerol
  • Concentration2.5 millimolar (mM) MgCl2
  • Concentration20 micromolar (µM) GDP
Note: please change salt as needed to maintain final salt of 80 mM NaCl








Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Take proper precautions while freezing grids.
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).
Preparing Sample
Preparing Sample
10m
10m
Spin down purified LRRK2 RCKW. Centrifigation10000 rcf, 4°C, 00:10:00 , (can be faster)
Leave protein on ice afterwards.

Note
For best results, reduce the amount of time between spinning and freezing samples.

Freezing Grids
Freezing Grids
20s
20s
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 Amount10 µL samples, good for freezing 2 grids. This is to minimize time spent outside of storage buffer, reducing aggregation.
Note
High concentrations favor dimers and trimers, while lower concentrations favor monomers.

(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.