Computational protocol: Structure based function-annotation of hypothetical protein MGG_01005 from Magnaporthe oryzae reveals it is the dynein light chain orthologue of dynlt1/3

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Protocol publication

[…] MGG_01005 and the MGG_01005-MoDyn1I2(117–150) complex were crystalized by sitting drop vapour diffusion using an Oryx4 crystallization robot (Douglas instruments Ltd). For initial trials, a mixture of 0.25 µL protein (8 mg/mL) and an equal volume of reservoir solution was equilibrated against 33 µL of reservoir solution at either 8 °C or 16 °C. Crystals of Modynlt1/3 appeared after two or three weeks from a reservoir condition of 2.0 M ammonium citrate. After optimization, the best crystals were obtained from drops containing 0.2 µL of protein (8 mg/mL) in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 0.2 µL 1.8 M ammonium citrate equilibrated at 16 °C. Crystals of selenium-derivatised Modynlt1/3 were obtained in the similar conditions as that of the wild type. For X-ray diffraction experiments, crystals were transferred to a reservoir solution containing 15–20% (v/v) glycerol as cryo-protectant, flash-cooled by plunging into liquid nitrogen and stored for later use.Modynlt1/3-IC117–150 complex crystals were initially obtained from a solution of 2.4 M ammonium sulphate, 0.1 M Bis-Tris (pH 5.5). After varying the concentration of protein and ammonium sulphate, and optimisation of the pH and the crystallisation temperature, larger and better-looking crystals were obtained from a solution of 2.0 M ammonium sulphate, 0.1 M Bis-Tris (pH 5.2) at 8 °C.Native and Selenium data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) and processed with HKL-3000. The data collection statistics are summarised in Table . The MGG_01005 structure was solved by single-wavelength anomalous dispersion (SAD) using the Autosol routine of PHENIX. The MGG_01005-MoDyn1I2(117–150) complex structure was solved by molecular replacement using the apo structure as a search model in Phaser. In both cases models were subsequently improved by manual rebuilding in Coot and further refined using PHENIX with TLS (Translation/Libration/Screw) restraints. Atomic coordinates and structure factors of MGG_01005 and MGG_01005-MoDyn1I2(117–150) complex were deposited in the PDB with the accession codes 5HXL and 5HYC respectively. Stereochemical validation of the model was performed with MolProbity. The refinement statistics and model quality parameters are detailed in Table . Dimer interface analysis was performed with the PISA server, (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Sequence alignment was performed with Clustal. Figures containing structures were generated with PyMOL (PyMOL Molecular Graphics system, Version 1.3 Schrodinger, LLC). […]

Pipeline specifications

Software tools HKL-3000, PHENIX, Coot, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Magnaporthe oryzae, Fungi
Chemicals Glutamic Acid