Computational protocol: Emodin targets the β-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter pylori: enzymatic inhibition assay with crystal structural and thermodynamic characterization

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Protocol publication

[…] HpFabZ crystallization was performed using hanging-drop vapor-diffusion method similar to our reported approach []. 1 μl of HpFabZ (~10 mg/ml) in crystallization buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl) was mixed with an equal volume of reservoir solution containing 2 M sodium formate, 0.1 M sodium acetate trihydrate at pH 3.6–5.6 and 2% w/v benzamidine-HCl. The mixture was equilibrated against 500 μl of the reservoir solution at 277K. When the dimensions of HpFabZ crystals grew up to 0.5 × 0.3 × 0.3 mm3 after 7 days, Emodin was added into the original drops to a final concentration of ~10 mM and soaked for 24 hours. The crystal was then picked up with a nylon loop and flash-cooled in liquid nitrogen. Data collection was performed at 100K using the original reservoir solution as cryoprotectant on an in-house R-Axis IV++ image-plate detector equipped with a Rigaku rotating-anode generator operated at 100 kV and 100 mA (λ = 1.5418 Å). Diffraction images were recorded by a Rigaku R-AXIS IV++ imaging-plate detector with an oscillation step of 1°. The data sets were integrated with MOSFLM [] and scaled with programs of the CCP4 suite []. Analysis of the diffraction data indicated that the crystal belongs to space group P212121. [...] HpFabZ-Emodin complex structure was solved by molecular replacement (MR) with the programs in CCP4 using the coordinate of native HpFabZ (PDB code is 2GLL) as the search model. Structure refinement was carried out using CNS standard protocols (energy minimization, water picking and B-factor refinement) []. Electron density interpretation and model building were performed by using the computer graphics program Coot []. The stereochemical quality of the structure models during the course of refinement and model building was evaluated with the program PROCHECK []. The coordinates and structure factor of the HpFabZ-Emodin complex structure have been deposited in the RCSB Protein Data Bank (PDB code is 3ED0). […]

Pipeline specifications

Software tools SHELX, iMosflm, CCP4, CNS, Coot, PROCHECK
Applications Small-angle scattering, Protein structure analysis
Organisms Helicobacter pylori
Chemicals Emodin