Computational protocol: Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles

Similar protocols

Protocol publication

[…] Chymotrypsinized and cross-linked L24C/R117C KcsA was crystallized in complex with Fab () by sitting drop vapor diffusion method at 20°C. The reservoir solution contained polyethylene glycol (PEG) 400 (23–25% wt/vol), MES (100 mM, pH 6.25–6.75), and magnesium acetate (25 mM). Cryoprotection was achieved by a stepwise increase of the PEG concentration in the reservoir at a rate of 5% per day until a final concentration of 40% was reached. Crystals were frozen and stored in liquid nitrogen.Diffraction data were collected at beamline 12-2 of the Stanford Synchrotron Radiation Light Source. Reflections were indexed, integrated, and scaled using XDS (). The structure was solved by molecular replacement using Phaser () and KcsA-Fab complex structure (PDB ID 1K4C) as search model (). The model was refined by iterative cycles of manual model building and refinement using COOT () and Phenix (), respectively. Structure validation was performed as described previously () to exclude model bias. [...] [1H,15N]HSQC NMR experiments were performed on a 600-MHz Varian Inova (Agilent Technologies) and a 600-MHz Bruker Avance (Bruker Corporation) equipped with cryogenic probes. Typical experiments ran for 18 h and were the mean of 512 scans with 128 real and complex points. Typical spectral widths for [1H,15N]HSQC were 12 ppm in the 1H dimension and 32 ppm in the 15N dimension. All NMR experiments were conducted at 45°C. Protein concentrations were 500 µM to 1 mM, and lipid concentration was limited to 100 mM. NMR data were processed with NMRPipe () and analyzed with NMRViewJ (). Graphs were produced with Prism (GraphPad Software). […]

Pipeline specifications

Software tools NMRPipe, NMRViewJ
Application NMR-based metabolomics
Chemicals Amino Acids, Cardiolipins, Phosphatidylcholines, Potassium