Oct 23, 2023
LRRK2-RCKW: MLi-2: E11 DARPin cryo-EM sample preparation
- 1University of California, San Diego
Protocol Citation: Marta Sanz Murillo, Andres Leschziner 2023. LRRK2-RCKW: MLi-2: E11 DARPin cryo-EM sample preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p17qgwz/v1
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: September 05, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 87407
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's: ASAP
Grant ID: ASAP-000519
Abstract
Protocol used to prepare LRRK2-RCKW: DARPin:MLi-2 complex and cryo-EM grid preparation.
Before start
Make
Protein purification and buffer exchange
Protein purification and buffer exchange
His6-Z-TEV-LRRK2-RCKW was expressed and purified as described in a previous protocol.
Protocol
NAME
LRRK1 expression and purificationCREATED BY
Robert Fagiewicz
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.
For the best result, keep protein on ice and reduce the amount of time between spinning
and 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
Expected result
The initial concentration range was 20-40 uM. The final concentration might be half of the initial one. Final volume might be 13-16 uL.
Thaw E11 DARPin and spin it down. Measure its concentration.
Dilute MLi-2 stock (diluted in 100% DMSO) to a desired concentration
Based on LRRK2-RCKW concentration, add the necessary volume to get a proportional ratio
LRRK2:DARPin:MLi-2 1:1.25:3 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.
E11 DARPin purification
E11 DARPin purification
E11 DARPin purified as described in the next protocol
cryo-EM sample preparation
cryo-EM sample preparation
We used UltraAuFoil Holey Gold 2/2 200 mesh grids and plasma cleaning them in the Solarus II (Gatan) using the QuantiFoil Au preset.
Dilute the sample to the desired concentration using the LRRK2 exchange buffer. We used 6 micromolar (µM).
Apply 3 to 3.5 microliters (µl) of sample and plunge freeze. We used a
Vitrobot (FEI) to blot away excess sample and plunge freeze in ethane liquid. (In
our case, we use 4 seconds as a time blot as 20 sec as a wait time and 4 as a blot force, but these
parameters are slightly different from one Vitrobot to another. I would try
with the Vitrobot parameters already tested in your machine first).
Store grids in liquid nitrogen until ready for imaging