Dear Sreetama, I would consider the possibility that this active site cysteine is involved in a mixed-disulfide with beta-mercaptoethanol, which is present at a considerable concentration in your protein buffer. The fact that the residual density in both the Fo-Fc and 2Fo-Fc maps actually increased beyond the modeled S-OH group after refinement and the features thereof, provide evidence for the likelihood of a mixed-disulfide with betaME.
best wishes Savvas ---- Savvas Savvides Unit for Structural Biology, L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html <http://www.lprobe.ugent.be/xray.html> > On 17 Jan 2015, at 19:22, sreetama das <somon_...@yahoo.co.in> wrote: > > Dear Users, > > I am solving a structure from x-ray diffraction data (1.62A resolution). > > The protein has a single cysteine residue (which is also the catalytic > residue), and it has a positive density on it (fig 1; R/Rfree = 16.88/19.94). > The positive density is retained upto 11.5 sigma level. > > Modelling with water retains the positive density (fig 2; R/Rfree = > 16.85/19.94) upto 5.2 sigma level. > > Modelling with CSO (S-hydroxycysteine, fig 3, R/Rfree = 16.82/ 19.81) > produces partial positive and negative densities, which are retained upto 5 > sigma. Moreover, after real-space refinement in coot followed by refinement > in refmac, the N-terminus of CSO is not bonded to the preceding residue, nor > is its C-terminus bonded to the succedding residue. > > All maps are contoured at 1sigma (2Fo-Fc map) and 3sigma (fo-fc map). > The protein preparation contained Tris buffer at pH 7.2, NaCl, glycerol and > beta-mercaptoethanol (2mM), while the crystallization condition contained > citric acid (pH 3.5) and ammonium sulfate. > > Please suggest how to interpret the data. > > thanking in advance, > sreetama > <coot_Cys-job12.png><coot_Cys+H2O_job10.png><coot_Cso-job11.png>
