Computational protocol: Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds

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Protocol publication

[…] We performed co-crystallization with organotin compounds and PPARγ-LBD by using the sitting-drop vapor-diffusion method at 277 K. Because the organotin compounds have poor aqueous solubility, an excess of each compound was added as powder to the protein solution and incubated for several hours to obtain the PPARγ-LBD/organotin complex. Insoluble compound was removed before crystallization by centrifugation followed by filtration through a 0.2-μm membrane filter. Crystals were obtained from drops derived from 1 μL of protein solution (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 5 mM DTT) mixed with an equal volume of crystallization buffer (100 mM Tris-HCl, pH 8.5 160 mM CH3COONH4, 19%–23% PEG4000). Diffraction data were collected at 100 K on beamline 6A or 17A of Photon Factory, KEK (Tsukuba, Japan), and beamline BL38B1 of SPring-8 (Hyogo, Japan), and were indexed, integrated, and scaled by using HKL2000. All structures were solved by use of the molecular replacement method using MOLREP from the CCP4 suite with the previously reported PPARγ-LBD structure (PDB 1PRG) as an initial search model. Structural refinement and the addition of water molecules were performed by using Coot and REFMAC5. The final structures were checked and validated by MolProbity. The atomic coordinates were deposited in the Protein Data Bank (PDB) as entry code 3WJ4 and 3WJ5 for PPARγ-LBD/TBT and PPARγ-LBD/TPT, respectively. The statistics for the diffraction data collection and structural refinement are shown in . […]

Pipeline specifications

Software tools Molrep, CCP4, Coot, REFMAC5, MolProbity
Application Protein structure analysis
Organisms Dipturus trachyderma
Diseases Hyperlipoproteinemia Type II
Chemicals Sulfur