Computational protocol: Phosphorylation-Dependent PIH1D1 Interactions Define Substrate Specificity of the R2TP Cochaperone Complex

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[…] Recombinant selenomethionine-substituted human PIH1D1 (51–180) was crystallized in complex with a diphosphopeptide (NH2-YAGSDpSDLDpSDDEFVPY-CONH) encompassing residues 483–497 of human TEL2 by sitting drop vapor diffusion with an IMPAX nano-dispensing robot. Crystals grew from a 8 mg/ml solution of complex in 20 mM Tris (pH 8.0), 150 mM NaCl, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) equilibrated well solution containing 32% w/v polyethylene glycol (PEG) 4000, 100 mM sodium acetate, and 100 mM Tris (pH 8.5) as precipitant, transferred to a cryo-protectant containing protein buffer and mother liquor supplemented with 20% v/v glycerol, and flash frozen in liquid nitrogen. All data were collected at Diamond Light Source. Images were indexed, integrated, and scaled with MOSFLM/SCALA (CCP4). Phases for the PIH1D1-pTEL2 complex were determined by single-wavelength anomalous diffraction and were of sufficient quality to enable a partial model of the two PIH1D1-pTEL2 complexes in the crystallographic asymmetric unit to be built automatically with Buccaneer (CCP4). All manual model building was carried out with Coot (), and structure refinement was carried out with Phenix (). The structure of unbound PIH1D1 (51–180) was crystallized by microseeding and vapor diffusion from 2% PEG 400 (v/v), 20% methoxy PEG 5000, and 0.1 M imidazole (pH 7.0). The structure was determined by molecular replacement using the final refined protein coordinates derived from the structure of the complex as a search model with PHASER () and refined against data extending to a 1.58 Å resolution. Crystallographic statistics are reported in . […]

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