5. sodium malonate, pH 7 8. 5. sodium phosphate. pH 7 8. 5. and a randomized screen obtained by randomly mix ing the above three precipitants Alisertib supplier with other additives. Seven differ ent crystal forms were identified from this comprehensive screen, as shown in Table 5 and Figure 4. Crystals for inhibitor soaking were grown in sitting drops by the vapor diffusion method using MK2. MK2 was added to 1. 5 L of reservoir solution and then the drop was sealed in vapor contact with 500 L of reservoir solution. Crystals grew to about 0. 2 mm in size in 3 days. For soaking, one MK2 crystal was added to 60 L of 1 mM inhibitor dissolved in mother liquor and incubated at 18 C overnight. Diffraction Testing and Structure Determination MK2 inhibitor complex crystals were harvested into a cry oprotectant solution using a fiber loop and flash cooled in liquid nitrogen.
Cystals were stored in liquid nitrogen until diffraction testing. X ray diffraction testing was conducted in house using a FR591 rotating anode generator with a MAR345 image plate detector Inhibitors,Modulators,Libraries and Osmic optics. A total of 535 crystals were tested, and over 80 crystals were selected for synchrotron data collection if diffraction reached at least 3. 5 resolu tion. Advanced Photon Source and National Synchrotron Light Source synchro tron beamlines were used primarily for data collection, although a few crystals were selected for in house data col lection. Diffraction data was processed Inhibitors,Modulators,Libraries with the HKL2000 pro gram suite. After determining the crystal orientation, the data were integrated with DENZO, scaled and merged with SCALEPACK, and placed on an absolute scale and reduced Inhibitors,Modulators,Libraries to structure factor amplitudes with TRUNCATE.
Five percent of the unique reflections were assigned, in a random fashion, to the free set, for calculation of the free R factor, the remaining 95% of reflec tions constituted the working set, for calculation of Inhibitors,Modulators,Libraries the R factor. The x ray diffraction data for a represent ative inhibitor soaked MK2 crystal are summa rized in Table 6. The CCP4 program suite was used to solve and refine the structure. The cross rotation function was calculated using MOLREP, using the apo MK2 structure reported Inhibitors,Modulators,Libraries previously as the search model. Initial sellckchem coordinates were generated based on the one solution apparent at 2. 9 resolution. Refinement began with rigid body refinement in REFMAC, resulting in an Rcryst of 37. 0% for all reflections with F 2. 0?F, 20 2. 9. Manual rebuilding of the model was conducted using the molecular graphics program O and examination of sigmaA weighted 2FO FC and FO FC electron density maps. Restrained refinement using REFMAC converged at an Rcryst of 22. 9%, 20 2. 9. The quality of the model was assessed with PROCHECK and WHATCHECK.