DYRK1A Kinase Structural Characterization

Recombinant DYRK1A was purified
as previously described^{60 (link)} and was treated
with TEV protease to remove the N-terminal His₆ tag. The
kinase at ∼13–15 mg/mL in 50 mM HEPES, pH 7.5, 500 mM
NaCl and 5 mM DTT was incubated with the inhibitors at 1 mM prior
to crystallization. Crystals were obtained using the sitting drop
vapor diffusion method at 4 °C using either 2 M ammonium sulfate,
0.2 M Na/K tartrate, 0.1 M citrate, pH 5.6 (for 5s and 5t), or 37% PEG 400, 0.2 M lithium sulfate, 0.1 M Tris, pH
8.8 (for 5j), as the reservoir solutions. Diffraction
data collected at Diamond Light Source, beamline I04-1, were processed
with XDS^{71 (link)} or mosflm^{72 (link)} and subsequently scaled with Scala from CCP4 suite.^{73 (link)} Structures were determined by molecular replacement
method using Phaser^{74 (link)} and the coordinates
of DYRK1A structure^{60 (link)} as a search model.
Iterative cycles of manual model building in COOT^{75 (link)} alternated with refinement using Refmac,^{76 (link)} and a TLS model calculated from TLSMD server^{77 (link)} was performed. The geometric correctness of
the final model was verified with MolProbity.^{78 (link)} Data collection and refinement statistics are summarized in Table 3. DYRK1A-ligand complexes (pdb id): DYRK1A-5j (4YLJ); DYRK1A-5s (4YLK); DYRK1A-5t (4YLL).