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[…] b'ts supplied by Hampton Research, and initial hits were further optimized. The best crystals of the FAAP20-UBZ\xe2\x80\xa2K63-Ub2 complex grew at 20\xc2\xb0C with the sitting drop vapor diffusion method by mixing 0.5 \xce\xbcl of a protein solution with an equal amount of a precipitant solution containing 1.8 M DL-malic acid (pH 7.0), 0.1 M glycine. For data collection, the crystals of the FAAP20-UBZ\xe2\x80\xa2K63-Ub2 complex were transferred to the precipitant solution containing saturated trehalose for cryoprotection. Cryoprotected crystals were flash frozen in liquid nitrogen., Diffraction data sets were collected at the beamline BL41XU in SPring-8 (Hyogo, Japan), and they were processed with the program HKL2000 [] and the CCP4 program suite []. The crystal of the FAAP20-UBZ\xe2\x80\xa2K63-Ub2 complex belongs to the space group P21, with unit cell dimensions of a = 59.8 \xc3\x85, b = 45.9 \xc3\x85, c = 172.9 \xc3\x85, \xce\xb2 = 98.1\xc2\xb0. The complex structures were determined by molecular replacement using the program MolRep []. The crystal structure of Ub (PDB code: 1ubq) was used as the search model. The atomic model of FAAP20-UBZ was built to fit 2F o-F c electron density map using the program COOT [] with careful inspection. Refinement was carried out using the program Refmac5 [] with iterative correction and refinement of the atomic model. The final model has excellent stereochemistry with an R free value of 23.5% at 1.90 \xc3\x85 resolution. Data collection and refinement statistics are shown in . All molecular graphics were prepared using the program PyMOL (DeLano Scientific; http://www.pymol.org)., GST-fused Ub and M1-Ub2 for SPR analyses were purified by Glutathione Sepharose FF and ResourceQ anion exchange columns (GE Healthcare). GST-fused K48-Ub2 was synthesized from Ub with an additional aspartate residue at its C-terminal extremity (D77 Ub) and GST-fused Ub carrying the K48R mutation. GST-fused K63-Ub2 was synthesized from D77 Ub and GST-fused K63R Ub. Ub conjugation reactions were performed as described previously []. Site-directed FAAP20-UBZ mutants were purified similarly to wild-type FAAP20-UBZ (see above). Experiments were carried out on a Biacore T200 instrument (GE Healthcare) equilibrated at 25\xc2\xb0C in HBS-P buffer including 10 mM HEPES-Na (pH 7.4), 150 mM NaCl ' […]

Pipeline specifications

Software tools CCP4, Coot, REFMAC5, PyMOL
Diseases Anemia, Bone Marrow Diseases, Anemia, Hypoplastic, Congenital, DNA Repair-Deficiency Disorders