Computational protocol: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

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Protocol publication

[…] Crystals of M. smegmatis ICD were obtained by vapour diffusion at 18 °C by equilibrating 2 or 4 μl hanging drops containing a 1:1 mixture of 60 mg ml−1 protein pre-incubated with 10 mM glyoxylate, 10 mM MnCl2, 10 mM isocitrate, and 10 mM NADP+ in crystallization buffer containing 0.1 M Hepes pH 7.5, 0.05 M MgCl2, and 28–34% polyethylene glycol monomethyl ether 550 (Qiagen) over a 500 μl reservoir of the same crystallization solution. Crystals were stabilized by soaking briefly in a cryoprotectant solution (25% w/v glycerol in crystallization buffer) and flash frozen in liquid nitrogen before data collection. Diffraction data to 2.8 Å (ICD incubated with glyoxylate, isocitrate, MgCl2 and NADP) were collected on X06DA of the Swiss Light Source (SLS, PSI, Villigen, Switzerland). Data were indexed, integrated and scaled with XDS. Phase determinations were carried out by molecular replacement using Phaser of the CCP4 Suite, using the published structure of AvICD (PDB: 1ITW) as a template. Structure figures were prepared with PyMOL (Molecular Graphics System, Version 1.5.0.1 Schrödinger, LLC). Crystallographic statistics are listed in . Coordinates and structure factors have been deposited in the Protein Data Bank (PDB accession code PDB: 4ZDA). [...] Protein preparation and grid generation. The raw PDB structure of the ICD was converted into an all atom receptor model, fully prepared for docking studies, using Protein Preparation Wizard in Maestro (Maestro v.9.8). Default parameters in the preparation process were used. The protein structural integrity was checked and missing side chains were built. The protonation and tautomeric states of the residues were adjusted to match a pH of 7.4. Energy minimization of the whole structure was performed using an OPLS2005 force field. The docking receptor grid was created using Glide Receptor Grid Generation (Glide v.9.8). The grid box was centered between the two domains of ICD, in a region that allowed the grid box to include the whole protein structure. All the possible binding sites were explored.Ligand preparation. The initial ligand file (sdf file, two-dimensional structure) was obtained from the Protein Data Bank (PDB). The glyoxylate was prepared for ligand docking using LigPrep from the Schrodinger suite. Default parameters in the ligand preparation process were used. A single low energy conformer with proper bond lengths and angles was generated from the two-dimensional structure. The protonation state was adjusted to match a pH of 7.4, tautomeric states were explored, and a final minimization step was performed. Glyoxylate was then docked inside the rigid 3D structure of the protein receptor (ICD), previously prepared. Glyoxylate was docked in the binding pocket and no other possible poses were found. An extra precision-docking revealed a binding energy of −6 kcal mol−1. […]

Pipeline specifications

Software tools XDS, CCP4, PyMOL, LigPrep
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Corynebacteriales, Escherichia coli
Diseases Glucosephosphate Dehydrogenase Deficiency, Infection, Tuberculosis
Chemicals Carbon, Fatty Acids