May 24, 2022

Public workspaceReconstituting LRRK2RCKW on Microtubules for cryo-EM studies

DOI
dx.doi.org/10.17504/protocols.io.bpnrmmd6
  • Mariusz Matyszewski1
  • 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.bpnrmmd6
Protocol Citation: Mariusz Matyszewski 2022. Reconstituting LRRK2RCKW on Microtubules for cryo-EM studies. protocols.io https://dx.doi.org/10.17504/protocols.io.bpnrmmd6
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: November 13, 2020
Last Modified: May 31, 2024
Protocol Integer ID: 44465
Keywords: LRRK2, microtubule interaction, Microtubule, ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
Abstract
This protocol contains a short instruction for reconstituting LRRK2RCKW on microtubules for cryo-EM studies with or without kinase inhibitors present.
Guidelines
Originally used without kinase inhibitors in:
CITATION
Deniston CK, Salogiannis J, Mathea S, Snead DM, Lahiri I, Matyszewski M, Donosa O, Watanabe R, Böhning J, Shiau AK, Knapp S, Villa E, Reck-Peterson SL, Leschziner AE (2020). Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.. Nature.
LINK
https://doi.org/10.1038/s41586-020-2673-2
Kinase inhibitor step was added for "Structural basis for Parkinson’s Disease-linked LRRK2’s binding to microtubules" by Snead, Matyszewski, Dickey et al.
Materials
Buffers needed:

Polymerization buffer
  • Concentration1 X BRB80
  • Concentration1 millimolar (mM) DTT
  • Concentration1 millimolar (mM) GTP
  • Concentration1 millimolar (mM) MgCl2
  • Concentration10 micromolar (µM) Taxol
  • Concentration10 % of either Glycerol (for low protofilament sized microtubules) or DMSO (for higher protofilament sizes) (DMSO was used in the original publication, and glycerol was used in Snead, Matyszewski, Dickey et al.)

LRRK2 Reaction buffer
  • Concentration20 millimolar (mM) HEPES pH 7.4
  • Concentration80 millimolar (mM) NaCl
  • Concentration0.5 millimolar (mM) TCEP
  • Concentration2.5 millimolar (mM) MgCl2
  • Concentration10 micromolar (µM) Taxol

Concentration1 X BRB80 (usually made as a 5X solution)
  • Concentration80 millimolar (mM) PIPES-KOH pH 6.8
  • Concentration1 millimolar (mM) MgCl2
  • Concentration1 millimolar (mM) EGTA
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Please take notice of the buffer preparation in section 'Materials'.
Instruction
Instruction
1h 0m 44s
1h 0m 44s
Add purified LRRK2RCKW and unpolymerized bovine tubulin dimer in a 2:1 molar ratio into the polymerization buffer (2 LRRK2RCKWs for each tubulin dimer). (Recommended total size: Amount10 µL )
Note
Note: Concentration of LRRK2RCKW has to be at least Concentration2.5 micromolar (µM) to see filaments occur. Has been tested with multiple variants.
For Snead, Matyszewski, Dickey et al, all filaments were formed at Concentration4.5 micromolar (µM) LRRK2RCKW and Concentration2.25 micromolar (µM) tubulin dimer.

Note
Note: NaCl concentration at this step should remain at around Concentration90 millimolar (mM) or less, often imposing a limit on LRRK2RCKW concentration allowed to be used. Higher salt concentration will prevent or reduce filament formation.

If incubating with LRRK2 kinase inhibitors, add those before adding tubulin to LRRK2RCKW and allow to incubate for at least Duration00:05:00 .


Note
In Snead, Matyszewski, Dickey et al, MLi-2 was added atConcentration5 micromolar (µM) (final concentration after adding tubulin) .


5m
Allow the mixture to polymerize at TemperatureRoom temperature for at least Duration01:00:00 .
1h
Prepare cryo-EM grids.
Recommended grids to use: Lacey Carbon on copper, 300 mesh, made by EMS. Glow discharged right before plunge freezing for Duration00:00:45 at 20 mA current.

45s
Dilute the sample 3-fold in the LRRK2 reaction buffer. (Recommended mixture: Amount4 µL sample + Amount8 µL LRRK2 buffer ).

Note
This step reduces glycerol to be within acceptable levels for cryo-EM.

This will reduce the effective concentration of components, but the dilution of LRRK2RCKW and tubulin might be non-linear due to filaments bundling to each other. The minimum concentration mentioned in Step 1 only applies to incubation step.

Apply diluted sample to grid and plunge freeze using your usual Vitrobot settings. (Ex. Amount4 µL sample , blotted for Duration00:00:04 at blot force 20 for our particular Vitrobot (FEI); conditions might vary from one machine to another).
4s
Citations
Deniston CK, Salogiannis J, Mathea S, Snead DM, Lahiri I, Matyszewski M, Donosa O, Watanabe R, Böhning J, Shiau AK, Knapp S, Villa E, Reck-Peterson SL, Leschziner AE. Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.
https://doi.org/10.1038/s41586-020-2673-2