Reconstituting LRRK2RCKW on Microtubules for cryo-EM studies

Abstract

This protocol contains a short instruction for reconstituting LRRK2^RCKW on microtubules for cryo-EM studies with or without kinase inhibitors present.

Before start

Please take notice of the buffer preparation in section 'Materials'.

Steps

Instruction

Add purified LRRK2^RCKW and unpolymerized bovine tubulin dimer in a 2:1 molar ratio into the polymerization buffer (2 LRRK2^RCKWs for each tubulin dimer). (Recommended total size: 10µL)

Note

Note: Concentration of LRRK2^RCKW has to be at least 2.5micromolar (µM) to see filaments occur. Has been tested with multiple variants.For Snead, Matyszewski, Dickey et al, all filaments were formed at 4.5micromolar (µM) LRRK2^RCKW and 2.25micromolar (µM) tubulin dimer.

Note

Note: NaCl concentration at this step should remain at around 90millimolar (mM) or less, often imposing a limit on LRRK2^RCKW concentration allowed to be used. Higher salt concentration will prevent or reduce filament formation.

1.1.

If incubating with LRRK2 kinase inhibitors, add those before adding tubulin to LRRK2^RCKW and allow to incubate for at least 0h 5m 0s.

Note

In Snead, Matyszewski, Dickey et al, MLi-2 was added at5micromolar (µM).

Allow the mixture to polymerize at Room temperature for at least 1h 0m 0s.

Prepare cryo-EM grids.

Recommended grids to use: Lacey Carbon on copper, 300 mesh, made by EMS. Glow discharged right before plunge freezing for 0h 0m 45s at 20 mA current.

Dilute the sample 3-fold in the LRRK2 reaction buffer. (Recommended mixture: 4µL + 8µL).

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 LRRK2^RCKW 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. 4µL, blotted for 0h 0m 4s at blot force 20 for our particular Vitrobot (FEI); conditions might vary from one machine to another).

Abstract

Before start

Steps

Instruction

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