Computational protocol: Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase

Similar protocols

Protocol publication

[…] Initial experiments for the screening of crystallization conditions was performed using the hanging drop method vapor-diffusion and incomplete factorial screens INDEX HT™ (Hampton research) JCSG-plus HT-96, PACT premierTM, ProPlex (Molecular dimensions). These studies provided the leads for optimization. All crystals for diffraction experiments were subsequently obtained by mixing 1 μL of the TbrHGPRT ligand complex and 1 μL of well solution. The protein-ligand complexes was prepared using 36 mg/mL, 28 mg/mL, 60 mg/mL and 28 mg/mL of enzyme in the presence of 3.2 mM (GMP), 3.2 mM (IMP), 4.6 mM (6), 4.6 mM (5) and 1.8 mM (3), respectively followed by incubation on ice for 10 min. The reservoir solution for the complexes with GMP (), IMP and 5 and 3 was 25% PEG 3350, 0.2 M lithium sulfate and 0.1 M Bis-Tris all within a pH range of 5 to 5.5. For 6 the well solution was 25% PEG 3350, 0.2 M sodium iodide, 0.1 M Bis-Tris propane, pH 6.5. Prior to cryocooling, all crystals were transferred to a cryoprotectant solution containing reservoir solution, 20% glycerol and 1–5 mM ligand. Crystals were subsequently transported to the Australian Synchrotron and robotically placed into the cryostream (100 K) on beamline MX1. X-ray data were collected remotely using BLU-ICE. Data were, integrated, scaled and merged using XDS. The structure of the complex with 3 was solved by molecular replacement using PHASER and the structure of TcrHGPRT (PDB code 1TC2) as the starting model. The starting models for the complexes with GMP, IMP, 5 and 6 were based upon the refined model of the complex with 3. Refinement and model building was performed with PHENIX 1.7.3 and COOT 0.8.2. […]

Pipeline specifications

Software tools XDS, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Trypanosoma brucei, Homo sapiens
Diseases Communicable Diseases, Lesch-Nyhan Syndrome, Trypanosomiasis, African
Chemicals Guanine, Atrial Natriuretic Factor, Nucleotides, Purines, Hypoxanthine