May 29, 2024

Public workspaceCryo-EM sample preparation for RCKW:DARPin complex

 Forked from Preparation of LRRK2 RCKW cryo-EM grids
DOI
dx.doi.org/10.17504/protocols.io.bp2l6224kgqe/v1
  • 1University of California, San Diego
Marta Sanz Murillo
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l6224kgqe/v1
Protocol Citation: Marta Sanz Murillo 2024. Cryo-EM sample preparation for RCKW:DARPin complex. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6224kgqe/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: January 31, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 100824
Keywords: cryo-EM, LRRK2, structural biology, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
Abstract
This is Leschziner's Lab protocol for making cryo-EM grids for RCKW:DARPin complex.
Materials
LRRK2 Buffer:
  • Concentration20 millimolar (mM) HEPES pH 7.4
  • Concentration150 millimolar (mM) NaCl
  • Concentration0.5 millimolar (mM) TCEP
  • Concentration5 % volume Glycerol
  • Concentration2.5 millimolar (mM) MgCl2
  • Concentration20 micromolar (µM) GDP
Note: please change salt as needed to maintain final salt of 150 mM NaCl








Safety warnings
Attention
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Take proper precautions while freezing grids.
Before start
Decide which protein concentration to use, and create the proper LRRK2 buffers in order to obtain the right salt concentration (150 mM NaCl).
Freezing Grids
Freezing Grids
20s
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 LRRK2 buffer. Make sure final salt is at 150 mM NaCl.
For best results, make Amount8 µL samples, good for freezing 2 grids. This is to minimize time spent outside of storage buffer, reducing aggregation.


Apply protein to grids and plunge freeze (3-4 uL)
We used a Vitrobot (FEI) to blot away excess sample and plunge freeze
Store grids in liquid nitrogen until ready for imaging.