LRRK2:DARPins complex preparation

Abstract

Protocol used to create LRRK2-RCKW: DARPin complex for cryo-EM grid preparation.

His6-Z-TEV-LRRK2-RCKW was expressed and purified as described in a previous protocol

Prepare LRRK2 buffer exchange: Keep it at 4ºC.

20 millimolar (mM) HEPES pH=7.4

150 millimolar (mM) NaCl

2.5 millimolar (mM) MgCl2

20 micromolar (µM) GDP

0.5 millimolar (mM) TCEP

Spin down purified LRRK2-RCKW (10000 rcf, 4°C, 10 minutes). Leave protein on ice afterward.

Note

For the best result, keep protein on ice and reduce the amount of time between spinningand freezing cryo-EM samples.

Exchange buffer using a spin desalting column (Zeba™ Spin Desalting Columns, 7K MWCO (Catalog number: 89877).

Spin down again the exchange buffer LRRK2-RCKW (10000 rcf, 4°C, 10 minutes) and measure the concentration. Leave protein on ice afterward

Thaw the DARPin protein of your interest (E11 or C12) and spin it down. Measure its concentration.

Based on LRRK2-RCKW concentration, add the necessary volume to get a proportional ratio

LRRK2:DARPin 1:1.25 and dilute to a final 10 micromolar (µM) LRRK2-RCKW concentration using exchange buffer (150 mM NaCl)

Incubate 10 minutes at RT. Afterward, keep it on ice until grid preparation.