Preparation of LRRK1 RCKW cryo-EM grids

Abstract

Protocol used to create LRRK1 RCKW grids for cryo-EM used in Snead, Matyszewski, Dickey et al.

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).

Steps

Preparing Sample

Spin down purified LRRK1 RCKW. 10000rcf,4°C

Leave protein on ice afterwards.

Note

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

Freezing Grids

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 10µL 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 2micromolar (µM) to 6micromolar (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 3.5µL 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.

Abstract

Before start

Steps

Preparing Sample

Freezing Grids

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