Sep 09, 2022

Public workspacePreparation of LRRK2 RCKW trimer cryo-EM grids

This protocol is a draft, published without a DOI.
  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
Mariusz Matyszewski
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Protocol Citation: Mariusz Matyszewski 2022. Preparation of LRRK2 RCKW trimer cryo-EM grids. protocols.io https://protocols.io/view/preparation-of-lrrk2-rckw-trimer-cryo-em-grids-brypm7vn
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: 46831
Keywords: cryo-EM, LRRK2, structural biology, ASAPCRN
Abstract
This protocol has been adapted from Deniston et al (https://doi.org/10.1038/s41586-020-2673-2)

Original protocol by Colin Deniston. Adapted to protocol.io by Mariusz Matyszewski.

This protocol describes how to create cryo-EM grids for LRRK2 RCKW. In particular, this protocol was used to obtain a high-resolution cryo-EM structure of the LRRK2 RCKW trimer, as well as lower resolution structures of RCKW monomers and dimers.
Guidelines
Newer protocol is available (see updated protocol here) with better results for monomeric protein. This is included mainly for archival reasons.
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








Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Take proper precautions while freezing grids.
Preparing Sample
Preparing Sample
Dialyze purified LRRK2 RCKW into the final LRRK2 buffer (see Materials)
Dilute the protein to the desired final concentration.

Note
High concentrations favor dimers and trimers, while lower concentrations favor monomers. For the published trimer structure, a final concentration of 4 uM was used (see Deniston et al).

(Optional) If adding inhibitors, add them after diluting to the final concentration and incubate on ice for Duration01:00:00 . If using Apo protein, then proceed immediately.
1h
Freezing Grids
Freezing Grids
20s
20s
Glow discharge grids.
We used UltrAuFoil Holey Gold 1.2/1.3 300 mesh grids and glow discharged them at 20 mA for Duration00:00:20 in a K100 instrument.

20s
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.