Computational protocol: Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

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Protocol publication

[…] Crystals of purified wild-type and the open-gate mutant yeast 20S proteasome were grown in hanging drops at room temperature, as described previously [], using a drop volume of 1.5 μl of 20S (20 mg/ml in 10 mM Tris/HCl, pH 7.5, and 1 mM EDTA) and 0.5 μl of reservoir solution containing 100 mM Mes, pH 7.0, 40 mM Mg(CH3COO)2, 15% MPD (2-methyl-2,4-pentanediol) and 10 mM EDTA. Proteasome–inhibitor complexes were generated by soaking crystals overnight in reservoir buffer containing 1 mM compound, 10% DMSO and 20% MPD followed by an additional 5 h in reservoir buffer containing 1 mM compound, 10% DMSO and 25% MPD before being flash-cooled in liquid nitrogen. Crystal data and refinement statistics are given in Supplementary Table S1 at Data were collected using the Structural GenomiX (SGX)-CAT beamline at the Advanced Photon Source (APS) synchrotron of the Argonne National Laboratory (U.S. Department of Energy Chicago, IL, U.S.A.) and processed using the programs iMOSFLM [] and SCALA []. Starting co-ordinates for each of the proteasome–inhibitor structures were taken from PDB entry 1G0U []. SigmaA-weighted Fo−Fc difference electron density for the β5 active site was used to model inhibitor co-ordinates, starting with a conformation generated using the small-molecule topology generator PRODRG []. Model building was performed using the program Coot [] and refinement was carried out using the CCP4i graphical user interface [] to the REFMAC program []. […]

Pipeline specifications

Software tools iMosflm, CCP4, PRODRG, Coot
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Neoplasms, Lymphoma, B-Cell