At the pro tein level, PADI2 was observed in both BT 474 an

[jy9202]

The pooled protein was concentrated and dialyzed overnight into 50 mM HEPES pH 7. 5, 100 mM NaCl, 5 mM DTT, 1 mM ADP. The resulting protein MAPK リン酸化反応 was pooled and used dir ectly for crystallization trials. Mouse Tyk2 crystallization Mouse Tyk2 protein was incubated with Compound 1 and concentrated to 10 mgmL. After 34 days, protein crystals grew using the vapor dif fusion method in sitting drop plates under the following condition 4. 34. 7 M ammonium formate, 100 mM Tris pH 8. 0. Crystals were subsequently used for soaking inhibitors of interest. Compound 2 was soaked into the Tyk2 crystals by adding 1 uM inhibitor to a 100 uL well of harvest mother liquor. Crystals were frozen from mother liquor solution containing 20% glycerol.

Mouse purchase MK-1775 Tyk2 structure determination X ray diffraction data from mouse Tyk2Compound 1 crystals were collected at the IMCA beamline 17ID at the Advanced Photon Source in Argonne, IL. The crys tals were maintained at 100 K with an Oxford Cryosys tems Cryostream cooler during data collection. A total of 180 frames were collected at an oscillation range of 1. 0. The data were processed with the HKL2000 suite of programs. After determining the crystal orientation, the data were integrated with DENZO, scaledmerged with SCALEPACK, placed on an absolute scale and reduced to structure factor amplitudes with TRUNCATE. Five percent of the unique reflections were assigned ran domly to the free set, for calculation of the free R factor. The remaining 95% of the reflections constituted the working set for calculation of the R factor.

The x ray diffraction data and refinement sta tistics are summarized in Table 2. A maximum likelihood molecular replacement solution was determined using the program PHASER. One Tyk2 monomer was located in the asymmetric unit, in the space オーダー MS-275 group P3121. The search model was a crystal structure of Jak2 reported previously. Coordinates were generated based on the molecu lar replacement solution. The refinement of the Tyk2 Compound 1 complex crystal structure began with the molecular replacement solution coordinates. Rigid body refinement was conducted by the program REFMAC in the CCP4 suite of programs, which resulted in the fol lowing statistics at 2. 6R 0. 39. Experimen tal Tyk2 and inhibitor electron density was observed. Manual building of Compound 1 into the density was attempted using the molecular graphics program O and examination of 2FoFc and FoFc electron density maps.

The refinement program REFMAC was used for it erative rounds of restrained refinement. Final rounds of refinement were conducted using AUTOBUSTER. which added water molecules to the final model, resulting in the following statistics R 0. 199. Final refinement statistics are shown in Table 3. The quality of all models was evaluated using COOT. The co crystal structure of Compound 2 complexed to Tyk2 was solved by molecular replacement using the Tyk2Compound 1 structure as a probe. An ori gin shift of was applied to match the Compound 1 coordinates. DETWIN was used with a twinning frac tion of 0. 24 to improve refinement statistics. Final rounds of refinement were conducted using AUTOBUSTER. Final refinement statistics are listed in Table 3.