We initially attempted crystallization of both the TRIB1 pseudokinase and pseudokinase plus C-terminal tail with free C/EBPα(53–75) peptide and the C/EBPα peptide fused to other proteins. We were able to eventually crystallize TRIB1(84–343) in fusion with C/EBPα(53–75), linked by a GSGSSGGPG linker. Initial crystals were grown by vapor diffusion, mixing fusion protein (~12.2 mg/ml) with 2.8 M sodium acetate (salt) and 0.1 M bis-tris propane (pH 7). Initial hits were refined using additive screening and soaking experiments, and crystals for data collection were grown in 2.8 M sodium acetate and 0.1 M bis-tris propane (pH 7) with 5% sodium citrate mother liquor diluted to 75 and 80% and drop ratios of 2:1 and 3:1. The crystals were cryoprotected in mother liquor supplemented with 25% glycerol. In attempts to improve diffraction, the crystal that eventually yielded the best resolution included 1 mM ATP in the cryoprotectant, but there was no electron density attributable to nucleotide in the final structure. Data were collected at the MX2 beamline of the Australian Synchrotron (Table 1) and processed using XDS and AIMLESS (46, 47). The structure was solved by molecular replacement in Phaser using the pseudokinase portion of PDB ID 5CEM as a model (48). Rebuilding was performed in Coot (49), and the structure was refined against diffraction data to a final resolution of 2.8 Å using REFMAC (50).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.