Mar 13, 2025

Public workspaceCo-crystallization of CLK3 with Ligand

Science Advances
DOI
https://dx.doi.org/10.17504/protocols.io.j8nlk8zj5l5r/v1
  • Nicolai D. Raig1,
  • Andreas Kramer1,
  • Stefan Knapp1
  • 1Structural Genomics Consortium (SGC)
Nicolai D. Raig
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlk8zj5l5r/v1
External link: https://doi.org/10.1126/sciadv.adt2050
Protocol Citation: Nicolai D. Raig, Andreas Kramer, Stefan Knapp 2025. Co-crystallization of CLK3 with Ligand. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8zj5l5r/v1
Manuscript citation:
Nicolai D Raig, Katherine J Surridge, Marta Sanz-Murillo, Verena Dederer, Andreas Krämer, Martin P Schwalm, Nicholas M Lattal, Lewis Elson, Deep Chatterjee, Sebastian Mathea, Thomas Hanke, Andres E Leschziner, Samara L Reck-Peterson, Stefan Knapp (2025) Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2.Science Advances doi: 10.1126/sciadv.adt2050
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: October 15, 2024
Last Modified: March 13, 2025
Protocol Integer ID: 109897
Keywords: clk3, ligand
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: 000519
Abstract

This is a step by step protocol, how to set up co-crystallization plates of CLK3 and compound, like it has been done for the PDB submission 9EZ3

Troubleshooting
Co-Crystallization of CLK3

  1. Incubate CLK3 protein kinase domain (~11 mg/mL) with compound of choice. 0.5-1 mM of compound if soluble
  2. Incubate the mixture on ice for 30 minutes.
  3. Prepare the crystallization firescreen around this condition: 15% PEG 6000, 0.1 M Bicine buffer pH 8.0 using 3 lenses 96 well plate
  4. Pipette 200-400 nL drops of the protein-reservoir mixture onto a sitting-drop vapor diffusion plate with robot or by hand (use 24 well plate), use protein:reservoir solution ratio 2:1 / 1:1 / 1:2
  5. seal plate and incubate at 4°C and incubate at 4°C
  6. Monitor crystal growth over time.
  7. Before fishing the crystals use Approx. 20-25% Ethylene Glycol as cryo protection
  8. Flash Freeze the crystal in Liquid N2
Protocol references