Purified Sxph was exchanged into a buffer of 10 mM NaCl and 10 mM HEPES (pH 7.4) and then concentrated to 65 mg ml−1 using a 50-kDa molecular weight cutoff Amicon Ultra centrifugal filter (Millipore) for crystallization screening by hanging drop vapor diffusion at 4°C using a 2:1 ratio of protein to screening solution. Apo-Sxph was crystallized from solution containing 0.1 M sodium cacodylate (pH 6.5), 5% PEG 8000, and 40% 2-methyl-2,4-pentanediol. Addition of 0.5% β-dodecyl maltoside or 10 mM sodium bromide to the crystallization solution further improved crystal quality of apo-Sxph. Crystals of the Sxph:STX complex were obtained by soaking STX (final concentration, 1 mM) into apo crystals for 5 hours before freezing. For cocrystallization, STX was added to Sxph in a molar ratio of 1.1:1 STX:Sxph. The sample was then incubated on ice for 1 hour before setting up crystallization. X-ray datasets for apo-Sxph and the Sxph:STX complex were collected at Advanced Light Source beamline 8.3.1 (Berkeley, CA) processed with XDS (58) and scaled and merged with Aimless (59). The apo-Sxph structure was determined by molecular replacement using Phaser from PHENIX (60) and the N-lobe of human serum transferrin (PDB: 1D4N) (61) as a search model. The resulting electron density map was further improved by rigid body refinement using phenix.refine and density modification using RESOLVE (62). The placed starting model was then subjected to model morphing in PHENIX using the prime-and-switch map generated from density modification (63). The morphed model thus allowed subsequent manual model building into the prime-and-switch map in COOT (64). Iterative model building, refinement, density modification, and model morphing allowed the apo-Sxph N-lobe and C1 domain to be built and refined (R-free of 31.7%) but left poor-quality electron density for the entire C2 domain. The feature-enhanced map (FEM) option (65) was applied in PHENIX to aid model building of the Sxph C2 domain. The C2 domain of rabbit serum transferrin (PDB: 1JNF) (41) was used as a starting model and placed into the FEM by rigid-body fitting. Subsequent model rebuilding and refinement were performed in COOT (64) and phenix.refine (60). The structure of the Sxph:STX complex was determined by molecular replacement using apo-Sxph as the search model in Phaser (60). Model building and refinement were carried out using COOT (64) and phenix.refine (60).

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