Computational protocol: Structure of Putrescine Aminotransferase from Escherichia coli Provides Insights into the Substrate Specificity among Class III Aminotransferases

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Protocol publication

[…] Native YgjG crystals were generated using the hanging-drop vapor-diffusion method at 22°C by mixing and equilibrating 2 µl each of the protein solution and a precipitant solution containing 0.1 mM n-dodecyl-N, N-dimethylglycine, 0.2 M sodium formate, 15% PEG 3350, and 0.1 M HEPES (pH 7.5) as described previously . The putrescine-bound structure was generated using the soaking method. For data collection, the crystals were briefly immersed in the same precipitant containing an additional 15% glycerol as a cryoprotectant and immediately flash-cooled in a 100 K nitrogen gas stream. Diffraction data were collected using an ADSC Q315r CCD detector on beamline 5C at Pohang Accelerator Laboratory (PAL), Pohang, Korea. A total of 240 images were collected with an oscillation of 1° and a crystal-to-detector distance of 200 mm. The crystal was exposed for 1 s per image. The data were indexed, integrated and scaled using the HKL-2000 software package . The YgjG structures were solved by using molecular replacement . The structure of acetylornithine aminotransferase (AcOAT) from Thermus thermophilus (PDB code 1VEF) was used as a search model to solve the YgjG crystal structures. Model building and refinement were performed using Coot and the Phenix package , respectively. The x-ray diffraction and structure refinement statistics are summarized in .Stereochemical analyses of the free and putrescine-bound structures were performed using the program PROCHECK . The atomic coordinates and structure factors are deposited in the Protein Data Bank with the codes 4UOY and 4UOX for the free and putrescine-bound YgjG, respectively. [...] The structures were superimposed using the Superpose program in the CCP4 suite . The dimer interface was analyzed by using the Protein Interfaces, Surfaces and Assemblies (PISA) server . Interactions between the YgjG and PLP cofactor residues were analyzed using the Contact program from the CCP4 suite . Structure-based multiple sequence alignments were performed using the programs MultAlin and ESPript . All structural figures were generated using Pymol (http://www.pymol.org). […]

Pipeline specifications

Software tools HKL-2000, Coot, PHENIX, PROCHECK, CCP4, MultAlin, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli
Chemicals Putrescine, Pyridoxal Phosphate