May 23, 2022

Public workspacePreparation of LRRK1 RCKW cryo-EM grids

DOI
dx.doi.org/10.17504/protocols.io.b3rqqm5w
  • 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.b3rqqm5w
Protocol Citation: Mariusz Matyszewski, David Snead 2022. Preparation of LRRK1 RCKW cryo-EM grids. protocols.io https://dx.doi.org/10.17504/protocols.io.b3rqqm5w
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 12, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 56848
Keywords: cryo-EM, LRRK2, structural biology, ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
Abstract
Protocol used to create LRRK1 RCKW grids for cryo-EM used in Snead, Matyszewski, Dickey et al.
Materials
LRRK1 Buffer:
  • Concentration20 millimolar (mM) HEPES pH 7.4
  • Concentration80 millimolar (mM) NaCl
  • Concentration0.5 millimolar (mM) TCEP
  • 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 LRRK1 buffers in order to obtain the right salt concentration (80 mM NaCl).
Preparing Sample
Preparing Sample
10m
10m
Spin down purified LRRK1 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 LRRK1 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
In our publication, we used concentrations ranging from Concentration2 micromolar (µM) to Concentration6 micromolar (µM) . Lower concentrations were used for samples collected on using a tilted stage.




Apply protein to grids and plunge freeze.
We used a Vitrobot (FEI) to blot away excess sample and plunge freeze

Note
Each Vitrobot is slightly different, so use your preferred settings. We used Amount3.5 µL of sample and a 4 second blot at force 20, but it is unlikely to work on other Vitrobots.


Store grids in liquid nitrogen until ready for imaging.