Computational protocol: Crystal Structure of Arginine Methyltransferase 6 from Trypanosoma brucei

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Protocol publication

[…] SAXS experiments were performed at NAL (Argonne National Laboratory). The data were acquired from three concentrations of FL-TbPRMT6: 1.0, 3.0, and 5.0 mg/ml in 30 mM Tris buffer (pH 8.0) with 200 mM NaCl and 1 mM EDTA. The data were analyzed using the ATSAS package following the standard procedures. After subtracting buffer scattering, the data curves obtained from the different concentrations were scaled and merged using PRIMUS . GNOM was employed to estimate the particle maximum dimension (Dmax) and calculate the pair distance distribution function (PDDF). The radius of gyration Rg of the protein was derived in real space using the PDDF. The solute molecular mass was evaluated by the SAXSMOW online tool . Models of chain-compatible dummy residues (DR) were constructed ab initio using the GASBOR program . The resolved X-ray structure of the TbPRMT6 dimer was superimposed onto the DR model by SUPCOMB . [...] The proteins were concentrated to approximately 15 mg/ml (determined photometrically) in 10 mM Tris (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 1 mM DTT. The protein-SAH complex was prepared by mixing the protein with a three-fold molecular excess of adenosyl-L-homocysteine (Sigma). All of the crystals were grown at 293K via the hanging drop method with the mother liquor containing 100 mM citrate (pH 5.5) and 20% PEG3000. The X-ray diffraction data for the crystals were collected on beamline 17U1 at the Shanghai Synchrotron Radiation Facility (SSRF). The data were processed and scaled with HKL2000. The statistics of the diffraction data are summarized in . The structure of SAH-TbPRMT6 was determined through the single-wavelength anomalous dispersion (SAD) phasing technique with the iodine anomalous signal using the phenix.solve program implemented in PHENIX –. The initial model was built automatically using the program Autobuild in PHENIX. Using the SAH-TbPRMT6 structure as the search model, the structures of apo-TbPRMT6 were determined through the molecular replacement method using the program MOLREP implemented in CCP4i –. All of the initial models were refined using the maximum likelihood method implemented in REFMAC5 as part of the CCP4i program suite and rebuilt interactively using the program COOT . During the later stage, the restrained positional and B-factor refinement was performed using the program phenix.refine during the refinement. The final models were evaluated with the programs MOLPROBITY and PROCHECK . The crystallographic parameters are listed in . All of the structures in the figures were prepared with PyMOL . […]

Pipeline specifications

Software tools ATSAS, GASBOR, PHENIX, Molrep, REFMAC5, Coot, MolProbity, PROCHECK, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Trypanosoma brucei, Bos taurus, Homo sapiens
Diseases Protein S Deficiency