Computational protocol: Structural and functional coupling of Hsp90- and Sgt1-centred multi-protein complexes

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Protocol publication

[…] HvHsp90-N and AtSgt1a-CS were combined in a 1:1 molar ratio in the presence of 5 mM ADP, incubated for 30 min and concentrated to 10 mg/ml by ultrafiltration. Initial multiple crystals were grown by vapour diffusion at 4°C against 26% w/v PEG4000, 100 mM Tris (pH 8.5) and 200 mM magnesium sulphate. Subsequent streak seeding into solutions of 16% w/v PEG4000, 100 mM Tris (pH 8.5) and 200 mM magnesium sulphate produced single thin plates. Crystals were harvested into reservoir solution with addition of glycerol (20% v/v) before flash cooling to 100 K. X-ray data were collected on beamline ID23 at the ESRF, Grenoble, from single crystals, and processed by using MOSFLM () and the CCP4 package (). Crystals had space group P212121, with three copies of the complex in the asymmetric unit. The thin plate crystals have a high solvent content (59% v/v) and only gave useful diffraction to 3.3 Å. The structure was solved by molecular replacement with Phaser () using ADP-bound yeast Hsp90-N (PDB code: 1AMW) and the solution structure of human Sgt1-CS domain (PDB code: 1RL1) as search models, built using COOT (), and refined with CNS in PHENIX (), using NCS restraints. Data collection and refinement parameters are given in . Crystallographic data have been deposited in the Protein Databank with code: 2JKI […]

Pipeline specifications

Software tools iMosflm, CCP4, Coot, CNS, PHENIX
Applications Small-angle scattering, Protein structure analysis
Organisms Saccharomyces cerevisiae