Computational protocol: Studies of a Ring-Cleaving Dioxygenase Illuminate the Role of Cholesterol Metabolism in the Pathogenesis of Mycobacterium tuberculosis

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Protocol publication

[…] X-ray data collections were performed under cryogenic conditions using an in-house rotating anode X-ray generator (CuKα radiation, λ = 1.542 Å) and at the Advanced Light Source (ALS, Beamline 8.2.2). Data were processed using HKL2000 . Molecular replacement was performed using PHASER and the structure of substrate-free BphC (PDB accession code 1HAN) with Fe and waters deleted as a search model. The highest scoring solution placed a dimer in the asymmetric unit, which was used as a starting model for re-building and structure refinement, which was performed using CNS (simulated annealing) and REFMAC in alternation with manual rebuilding using COOT .For the HsaC:DHSA complexes, difference Fourier electron density maps revealed additional density within the active site consistent with a bound DHSA. The diffraction data and properties of the refined model are characterized in . A model for the substrate was established using the PRODRG server. Electron density maps were calculated with the CCP4 suite (FFT function). Structural figures and graphical rendering were made by using PYMOL . The final model of HsaC:DHSA contains a dimer of HsaC covering 299/298 residues of each chain, two DHSA molecules, and 485 water molecules. The final model of substrate-free HsaC contains a dimer of HsaC covering 295 residues (2–296) of each chain, one tartrate and 712 water molecules. The coordinates for HsaC:DHSA and HsaC alone were deposited in the Protein Data Bank ( with accession codes 2ZI8 and 2ZYQ, respectively. […]

Pipeline specifications

Software tools CNS, Coot, PRODRG, CCP4, PyMOL
Applications Drug design, Protein structure analysis
Organisms Cavia porcellus, Mus musculus, Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv
Diseases Infection, Tuberculosis, Tuberculosis, Meningeal, Drug-Related Side Effects and Adverse Reactions
Chemicals Catechols, Cholesterol, Iron