Computational protocol: Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen

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Protocol publication

[…] The purification of Ag85s and mutants were carried out . Briefly, the plasmids encoding the genes of interest were used to transform E. coli T7 express cells (New England BioLabs). Bacterial cells were cultured at 37 °C in Luria Broth (Research Products International) supplemented with kanamycin (Gold Biotechnology) and chloramphenicol (Alfa Aesar). At OD600nm 0.6, the temperature was decreased to 16 °C and protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (Gold Biotechnology). The bacterial cells were harvested by centrifugation after 24 to 36 hours induction and the pelleted cells were resuspended into 20 mM Tris pH 8.0, 25 mM imidazole and 5 mM β-mercaptoethanol (Nickel affinity binding buffer). Cell lysis was performed by addition of lysozyme (Sigma Aldrich) and a sonication step (Sonicator 3000, Misonix). Then, nickel affinity and anion exchange purifications were carried out . The protein was concentrated using ammonium sulfate precipitation (2.6 M). The precipitated protein was resuspended and dialyzed into either crystallization buffer (10 mM Tris pH 7.5, 2 mM EDTA and 1 mM dithiothreitol [DTT]) or assay buffer (50 mM sodium phosphate pH 7.5, 1 mM dithiothreitol).The enzyme concentration was determined using absorbance spectroscopy at 280 nm. The ExPASy proteomics server, specifically the ProtParam function, was used to calculate the extinction coefficients of Ag85C and its different mutants . Recombinant Mtb Ag85A and B were purified using the same protocol. [...] Diffraction data were indexed, integrated and scaled using HKL2000 . EPMR (Evolutionary Program for Molecular Replacement) was used to perform the molecular replacement for each data set using PDB 1DQZ (Ag85C WT) as a model. Rigid body refinement, simulated annealing, positional, and B-factor refinement was carried out using PHENIX . Manual refinement was performed using COOT . The final refined model results in a Ramachandran plot with 97.0% of residues in the favored region and 3.0 % in the allowed for the Ag85C-EBS structure. In the Ag85C-C209S structure, the favored and allowed values are 97.5% and 2.1%, respectively. […]

Pipeline specifications

Software tools ProtParam, PHENIX, Coot
Databases ExPASy
Applications Protein structure analysis, Protein physicochemical analysis
Organisms Mycobacterium tuberculosis, Mycobacterium
Chemicals Cysteine, Hydrogen