Computational protocol: Ligand complex structures of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change

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Protocol publication

[…] N‐terminally His‐tagged recombinant l‐AAO/MOG protein was expressed in Escherichia coli and purified as described previously . The purified protein was dialyzed against 20 mm HEPES–NaOH (pH 7.0) and concentrated using Amicon centrifugal filters (Millipore, Billerica, MA, USA) with a 30 kDa cutoff membrane to 20 mg·mL−1 for crystallization. Protein crystallization was performed by sitting‐drop vapor diffusion method at 20 °C by mixing 1 μL of protein solution and 1 μL of reservoir solution. The reproducibility of the crystallization conditions described in the previous report (8% PEG4000 and 0.1 m sodium acetate, pH 4.6) was not high enough for extensive soaking experiments. After crystallization screening from scratch, a new condition with high reproducibility was found. Native l‐AAO/MOG crystals were obtained using reservoir solution consisting of 20% of PEG3350 and 0.15 m dl‐malic acid, pH 7.0. Complex crystals were prepared by soaking the crystals for 10–30 s in a reservoir solution supplemented with 20% glycerol and 10 mm l‐Lys, 20 mm l‐Orn, or 20 mm l‐Arg. Crystals were flash‐cooled at 100 K in a stream of nitrogen gas. Diffraction data were collected at beamline BL5A at the Photon Factory, High Energy Accelerator Research Organization (KEK; Tsukuba, Japan) at a wavelength of 1.000 Å. The diffraction images were processed using hkl2000 (l‐Lys complex) and xds (l‐Orn and l‐Arg complex) . The initial phases of the ligand complex structures were solved by molecular replacement using the native l‐AAO/MOG structure (PDB ID: 3WE0) as a search model. Manual model rebuilding and refinement were achieved using coot , refmac5 , and phenix . The resultant F o–F c and 2F o–F c maps yielded an electron density that corresponded to the soaked substrate. Figures were prepared using Open Source pymol (Schrödinger, LLC, New York, NY, USA), cuemol (, and caver 3.0 . […]

Pipeline specifications

Software tools XDS, Coot, REFMAC5, PHENIX, PyMOL, CueMol, CAVER
Applications Small-angle scattering, Protein structure analysis
Chemicals Amino Acids