Mar 12, 2025

Public workspaceCo-crystallization of CLK3 with Ligand

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8zj5l5r/v1
  • 1Structural Genomics Consortium (SGC)
Nicolai D. Raig
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk8zj5l5r/v1
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
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 12, 2025
Protocol Integer ID: 109897
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

Co-Crystallization of CLK3
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