Computational protocol: Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition

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Protocol publication

[…] X-ray diffraction data from crystals of wild-type zNt-FDH were first collected at 110 K using a CCD detector (Q210R, ADSC) at a wavelength of 1.0 Å on the Taiwan-contracted beamline BL12B2 at SPring-8, Japan. 180° of rotation was measured with 1.0° oscillation, an exposure duration of 15 s and a crystal-to-detector distance of 180 mm at 110 K using a cryosystem (X-Stream, Rigaku/MSC). All other X-ray diffraction data were collected at 110 K on beamlines BL13B1, BL13C1 and BL15A1 equipped with CCD detectors (ADSC Q315 and Rayonix MX300HE) at the National Synchrotron Radiation Research Center (NSRRC), Taiwan and on BL12B2 and BL44XU with a CCD detector (Rayonix MX225HE) at SPring-8. All data sets were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997). Details of the data statistics are given in Table 1. [...] The structure of wild-type zNt-FDH was solved by molecular replacement using the structure of Nt-FDH from rat (PDB entry 1s3i) as the search model (sequence identity 73%; Chumanevich et al., 2004). Throughout the refinement, a random selection (5%) of the data was set aside as a ‘free data set’ and the model was refined against the remaining data as a working data set (Brünger, 1992). Model building and refinement were performed using Coot, REFMAC5 and PHENIX (Emsley et al., 2010; Vagin et al., 2004; Adams et al., 2010). Several cycles of model building using Coot and refinement with the individual atomic displacement parameters, TLS (translation, libration, screw) and target weight options were used to improve the quality and completeness of the structures. The refinement proceeded through another cycle of individual B-factor refinement and water assignment with Coot and PHENIX. The refinement converged to a final R work of 18.0% (R free of 20.7%) at a resolution of 1.75 Å. The structures of the wild-type complexes and the apo form and complex of the Y200A mutant were determined by molecular replacement using the determined wild-type structure as the search model with MOLREP (Vagin & Teplyakov, 2010). The structures were refined using procedures similar to those described above. The correctness of the stereochemistry of the models was verified with MolProbity (Chen et al., 2010). The calculations of root-mean-square deviations from ideality for bonds, angles and dihedral and improper angles showed satisfactory stereochemistry. All crystallographic data and refinement statistics are summarized in Table 1. The structures have been deposited in the PDB ( as entries 4ts4, 4tt8, 4qpd, 4qpc, 4tts and 4r8v. The figures were generated using PyMOL ( Table 1 shows details of the refinement statistics. […]

Pipeline specifications

Software tools HKL-2000, Coot, REFMAC5, PHENIX, Molrep, MolProbity, PyMOL
Databases wwPDB
Applications Small-angle scattering, Protein structure analysis
Organisms Danio rerio
Diseases Glucosephosphate Dehydrogenase Deficiency
Chemicals Folic Acid