Computational protocol: Identification and Structure–Function Analysisof Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

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Protocol publication

[…] Human GRK2 and Gβγ were mixed in a 1:1 ratio and concentrated to a final total protein concentration of approximately 10 mg mL–1 in the presence of 500 μM GSK180736A (from a 10 mM stock in DMSO) and 2 mM MgCl2. Crystals were obtained via hanging drop vapor diffusion using 0.8 μL of protein solution mixed with 0.8 μL of well solution (1 mL), which consisted of 1.2 M NaCl, 100 mM MES, pH 6.75, and 15% (w/v) PEG3350. Crystals appeared in approximately 1 week and continued to grow in size for several weeks. During harvesting, the crystals were cryoprotected via the addition of 25% (v/v) ethylene glycol to the harvested crystals prior to flash freezing in liquid N2. Diffraction data was collected at the Advanced Photon Source (APS) on LS-CAT beamline ID-F at a wavelength of 0.9787 Å. Data was collected from four sweeps collected from a single crystal. Indexing, integration, and scaling were performed with HKL2000. Initial phasing was performed via molecular replacement solution using PDB entry 3V5W as a search model in PHASER.,, Refinement was performed with the REFMAC5 module of CCP4 alternating with model building in Coot.− The final model was verified with MolProbity. [...] Buried surface area calculations were performed with the AREAIMOL extension of CCP4., Structural similarity calculations were performed via the PDBeFold server, and domain rotations calculations were performed on isolated kinase domains via the DynDom server., […]

Pipeline specifications

Software tools REFMAC5, CCP4, Coot, MolProbity, PDBeFold, DynDom
Application Protein structure analysis
Diseases Heart Failure, Parkinson Disease
Chemicals Paroxetine