2.3. Crystallization and X-ray structure determination of EH3 complexed with methyl-(R/S)-2-phenylpropanoate

The crystallization conditions reported for the native protein were optimized by adjusting the protein and precipitant concentrations. The best crystals were grown by using 1 µl of EH_3S192A (20–60 mg ml⁻¹ in 40 mM HEPES (pH 7) and 100 mM NaCl) and 0.5 µl of precipitant solution (28–29% PEG3000, 0.1 M Bis-tris (pH 6.5), and 0.2 M MgCl₂∙6H₂O). The complexes were obtained by soaking thin plate-shaped crystals of EH_3S192A in mother liquor supplemented with 10–20 mM methyl-(S/R)-2-phenylpropanoate for 1–3 h. For data collection, crystals were transferred to cryoprotectant solutions consisting of mother liquor plus 20–23% (v/v) glycerol before being cooled in liquid nitrogen. Diffraction data were collected using synchrotron radiation on the XALOC beamline at ALBA (Cerdanyola del Vallés, Spain). Diffraction images were processed with XDS [40] and merged using AIMLESS [41] from the CCP4 package [42]. Both crystals were indexed in the C2 space group, with two molecules in the asymmetric unit and 40% solvent content within the unit cell. The data collection statistics are given in Table S1.

The structure of the complex was solved by difference Fourier synthesis using the coordinates of the EH₃ native crystals (PDB ID: 6SXP). Crystallographic refinement was performed using the program REFMAC [43] within the CCP4 suite with local noncrystallographic symmetry (NCS). The free R-factor was calculated using a subset of 5% randomly selected structure-factor amplitudes that were excluded from the automated refinement. At the later stages, ligands were manually built into the electron density maps with Coot [44], and water molecules were included in the model, which, when combined with more rounds of restrained refinement, reached the R factors listed in Table S1. For methyl-(R/S)-2-phenylpropanoate, which is not present in the Protein Data Bank, a model was built using MacPyMOLX11Hybrid (the PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC). The model was used to automatically generate coordinates and molecular topologies with eLBOW [45], which is suitable for REFMAC refinement. The figures were generated with PyMOL. The crystallographic statistics of EH3_S192A complexed with methyl-(R/S)-2-phenylpropanoate are listed in Table S1.