Crystallization and x-ray structure determination

Prior to crystallization, NfCYP51 was diluted to 0.5 mM by mixing with 0.6 mM inhibitor added to the desired volume of water from the 10 mM or 100 mM stock solutions in DMSO, depending on compound solubility. Screening of crystallization conditions for each inhibitor complex was performed using commercial high-throughput screening kits available in deep-well format (Hampton Research or Qiagen), a nanoliter drop-setting Mosquito robot (TTP LabTech) operating with 96-well plates, and a hanging drop crystallization protocol. For diffraction quality, crystals were further optimized in 96-well plates configured using the Dragonfly robot (TTP LabTech) and the Designer software (TTP LabTech). All crystals were harvested from the narrow grid of crystallization conditions: 30–33% PEG MME 550, 30 mM CaCl₂, 0–4% Jeffamine M-600, 0.1 M bis-Tris propane, pH 7.1–7.5.

Diffraction data were collected at 100–110 K at beamline 8.3.1, Advanced Light Source, Lawrence Berkeley National Laboratory, USA. Data indexing, integration, and scaling were conducted using XDS.[74] The high-resolution crystal structure of the NfCYP51-posaconazole complex was determined by molecular replacement using as a search model T. cruzi CYP51, PDB ID 2X2N. The initial model was built using the BUCCANEER[75, 76] and COOT[77] programs. Refinement was performed by using REFMAC5 software.[76, 78] The newly determined crystal structure (PDB ID 5TL8) was subsequently used for other NfCYP51-conazole complexes reported in this work. Data collection and refinement statistics are shown in Table 4.

The atomic coordinates and structure factors (5TL8, 6AY4, 6AY6, 6AYB and 6AYC) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/)