Computational protocol: Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis

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Protocol publication

[…] Crystal diffraction data of apo-ACP and FabZ-ACP complex were collected at beamline BL19U1 at the National Center for Protein Sciences Shanghai and Shanghai Synchrotron Radiation Facility, China. Significant decay of the complex crystals during data collection caused the resolution cut off at 2.55 Å. While the crystal diffraction data of holo-ACP were collected at 100 K using CuKα X-ray with a Rigaku R-AXIS IV++ image plate. The data were further processed using HKL2000 software package and CrystalClear software. The structures were solved by MR with the published structure of apo-EcACP (PDB code: 1T8K) and HpFabZ (PDB code: 2GLL) as search models, and refined by using programs Phenix (complex) and CNS (apo and holo-ACP). The crystallographic statistics are summarized in . Electron density interpretation and model building were performed using the computer graphics program Coot. Final structures were visualized by PyMol software. [...] FabZ-ACP complex purified from gel-filtration in the solution containing 20 mM HEPES, pH 8.0, 150 mM NaCl and 10 mM DTT was used in the Synchrotron SAXS experiment. The measurement was performed in a vacuum cuvette with exposure time of 1 s to diminish the parasitic scattering under wavelength 1.03 Å at National Center for Protein Sciences Shanghai (NCPSS) beamline BL19U2, China. The scattering was recorded in the range of the momentum transfer 0.015 < s < 0.420/Å, where s = (4π × sinθ)/λ and 2θ is the scattering angle. SAXS data were subsequently processed and analyzed by using the ATSAS program suite. Briefly, program PRIMUS was employed to process the experimental scattering data and correct the solvent background scattering. Program CRYSOL was used to generate the theoretical scattering from the FabZ-ACP complex crystal structure. Program GNOM was employed to calculate the distance distribution function p(r) ( and ). Subsequently, programs DAMMIN, GASBOR and DAMAVER were used to reconstruct the low-resolution shapes to a dummy atom particle model, and superpose to the crystal structure (). The figure was generated by using GraphPad Prism 5 and PyMol software. […]

Pipeline specifications

Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Fatty Acids