Computational protocol: Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity

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Protocol publication

[…] The GST-cleaved MPK6, MPK6Δ1–28, and MPK6Δ1–33 proteins were concentrated to 10 mg/ml. The crystals were grown in drops composed of 1 μl of protein solution, 1 μl of reservoir solution containing 100 mM citrate sodium, pH 6.2, 10–12% PEG20000 (Hampton Research), using the sitting-drop vapour-diffusion method at 22 °C. After streak seeding, the shape of crystals changed from tiny needle clusters to long, large rods. Only crystals of MPK6(Δ1–28) were obtained. The crystal was mounted in 50 μl of reservoir solution with 10% DMSO and air dehydrated for 5 min. The crystal was then transferred to 50 μl of reservoir solution containing 20% DMSO and air dried for 5 min. The crystals were flash-frozen in liquid nitrogen without any other cryoprotectant.The diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) at beam line BL17U1 using a CCD detector with an exposure time of 1 s per image at a crystal to detector distance of 380 mm. The data were processed using HKL2000. The crystal structure was solved using the PHASER program in the CCP4 program suite. The atomic coordinates of human ERK2 (PDB code 1ERK) were used as the starting search model. The model was further refined with either PHENIX or the CCP4 program. The statistics on data processing and structure refinement are listed in . The structure figures were created in PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC). […]

Pipeline specifications

Software tools CCP4, PHENIX, PyMOL
Application Protein structure analysis
Organisms Arabidopsis thaliana, Saccharomyces cerevisiae
Chemicals Glycine