NMR structure of the Saccharomyces cerevisiae Ste50 SAM domain
Saccharomyces cerevisiae Ste50 binds the MAPKKK Ste11 through a head-to-tail SAM domain interaction.
this protein fragment was cloned into the NdeI and BamHI sites of pET15b creating an aminoterminal hexahistidine tagged protein with an intervening thrombin protease cleavage site. After thrombin treatment, the non-native residues GSH remained amino terminal to the native Ste50 sequence
similar to Grimshaw SJ et al. (2004). J.Biol.Chem. 279(3) :2192-2201
This structure was determined using standard 3D heteronuclear techniques
Experimental observations: 615 intermolecular NOE distance restraints, 328 short range NOE distance restraints, 163 medium range NOE distance restraints, 149 long range NOE restraints, 42 pairs of hydrogen bond distance restraints and 69 pairs of phi/psi dihedral angle restraints. An initial ensemble of 500 structures were calculated with CYANA 2.0. The top 25 structures with minimum restraint violations were refined in water using XPLOR-NIH and a protocol by C. Spronk. All 25 water refined structures had no NOE violations > 0.5 A and no dihedral violations > 5 degrees. For residues 35-100, the backbone RMSD of the ensemble is 0.75 +/- 0.14 A. Residues 28-32 and 101-107 are disordered.
0.8 mM Ste50 SAM U-15N,13C, 20 mM sodium phosphate buffer, pH 7.8, 150 mM sodium chloride, 0.02 % sodium azide, 90% H2O, 10% D2O
all helix protein, SAM domain, CELL CYCLE