Computational protocol: Functional Complementation and Genetic Deletion Studies of KirBac Channels

Similar protocols

Protocol publication

[…] Homology models and molecular dynamics (MD) simulations were done as has been described before (). Briefly, a homology model of wild-type KirBac6.1 (residues 21–303) was built using Modeler 9v2 using KirBac3.1 crystal structure (Protein Data Bank (PDB): 1XL6) () as a template. Sequence alignments were made using ClustalW2. The selectivity filter was populated with K+ ions at S0, S2, and S4 and interspersed with waters using crystal structure coordinates. The cavity was flooded using packages Voidoo and Flood. The model was inserted into an explicit, pre-equilibrated phosphatidylglycerol:palmitoyl-oleoyl-phosphatidylglycerol bilayer in water, and additional waters were added to accommodate the KirBac6.1 C terminus (box size = 10 × 10 × 12 nm). The protein was optimally positioned in the bilayer, clashing (<2 Å) lipids and waters were removed, and KCl concentration was adjusted to 100 mm. MD simulations were carried out using GROMACS v3.3.3 and GROMOS forcefield. Prior to simulation, energy minimization was carried out followed by incremental (5 ps, 10–20-K steps) warming from 80 to 310 K (37 °C) and a 50-ps equilibration step, both with lipid headgroups restrained. The MD run was 15.2 ns.Mutant models were created by introducing the mutation (either W48R or G137D) into all four subunits using PyMOL version 0.99 after energy minimization. Further energy minimization, warming, equilibration, and MD simulation used identical parameters to the WT model. Root mean squared deviation analysis showed all three models to be stable between 5 and 15.2 ns of simulation; only data from this period were used in other analyses. […]

Pipeline specifications

Software tools Clustal W, VOIDOO, GROMACS, PyMOL
Application Protein structure analysis
Organisms Saccharomyces cerevisiae, Escherichia coli, Homo sapiens
Chemicals Potassium