Crystals were only obtained in the presence of 10 mM 2′-FL or 3-FL by vapor diffusion in hanging or sitting drops and grew for 1 week at room temperature at a 1:1 ratio of the native FL2-BP or the selenomethionine (SeMet)–labeled protein [100 mg/ml in 10 mM MES (pH 6.5) and 150 mM NaCl] and reservoir solution [100 mM MES (pH 6.5), 25% polyethylene glycol 500 monomethyl ether (PEG-500MME), and 10 mM ZnSO4]. The SeMet-labeled protein was expressed as previously described (35) and purified similarly as described for WT protein as above. The crystals were flash-frozen in liquid nitrogen without cryoprotectant in nylon loops. Diffraction data were collected to a maximum resolution of 2.1 Å for SeMet-labeled FL2-BP complexed with 2′-FL, 1.3 Å for FL2-BP complexed with 2′-FL, and 1.4 Å for FL2-BP complexed with 3-FL at the European Synchrotron Radiation Facility (ESRF) beamlines, Grenoble, France. All datasets were processed with XDS (36). The SeMet-labeled FL2-BP crystal diffracted to ~1 Å, but we were not able to process it beyond 2.1 Å. The structure was solved in the rhombohedral space group R3 in hexagonal setting (H3) using single-wavelength anomalous diffraction, with the experimental phase information obtained from data collected at the selenium K-edge. The program Phenix.AutoSol (37, 38) identified all selenium atoms in the 13 possible SeMet residues in the FL2-BP. An initial partial model was obtained with Phenix.AutoBuild (39). Further corrections and model building using the program Coot (40) resulted in a complete model, which was used in molecular replacement to solve the structure of FL2-BP in complex with 2′-FL. The models were refined using phenix.refine (41), randomly setting aside 5% of the reflections using identical R-free sets for all refinements. Molecular replacement with the protein part of FL2-BP was used to solve the 3-FL–complexed structure. Ligand molecules were included manually using Coot after the protein atoms were built, and water molecules were added. The overall quality of all models was checked using MolProbity (37). Data collection and refinement statistics are shown in table S1. The two complexes are very similar in conformation and superposition of the individual models results in pairwise overall RMSD of 0.2 Å between aligned Cα atoms. The PyMOL Molecular Graphics System (version, Schrödinger, New York, NY, USA) was used to explore the models and for molecular graphics.

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