Re: [ccp4bb] additional density on cysteine residue
Don't forget to check the anomalous difference Fourier - this may fix any S atoms - the resolution is god enough Eleanor On 18 January 2015 at 01:12, Robert Stroud str...@msg.ucsf.edu wrote: I suspect it may be a reaction with your reducing agent. What did you use either in the preparation, or in the crystallization. If you didn’t have reducing agent it probably oxidized to sulfuric acid. You should figure it out with difference maps and maybe mass spec also. bob On Jan 17, 2015, at 4:10 PM, sreetama das somon_...@yahoo.co.in wrote: Hi, Thanks for the quick replies. I modelled in CME and refined. However, I get a blob of negative density around the S-S bond, which is retained up to 5 sigma. Does it mean it is not CME ? Thanks regards, sreetama On Sunday, 18 January 2015 12:41 AM, Roger Rowlett rrowl...@colgate.edu wrote: If CSO does not account for the density -- the SO bond should be about 1.8 A IIRC -- a possibility is an adventitious metal ion. Roger Rowlett On Jan 17, 2015 1:25 PM, 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_CME.png all the best! Bob
Re: [ccp4bb] additional density on cysteine residue
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.pngcoot_Cys+H2O_job10.pngcoot_Cso-job11.png
Re: [ccp4bb] additional density on cysteine residue
Hi Sreetama, The water S-gamma distance made me think that it might be a cysteine beta-mercaptoethanol adduct. Try building CME instead of CSO. Cheers, Robbie -Oorspronkelijk bericht- Van: sreetama das somon_...@yahoo.co.in Verzonden: 17-1-2015 19:27 Aan: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK Onderwerp: [ccp4bb] additional density on cysteine residue 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
Re: [ccp4bb] additional density on cysteine residue
Maybe somehow do partial cys partial cme, refine occupancies—is this possible in refmac? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of sreetama das Sent: Saturday, January 17, 2015 3:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] additional density on cysteine residue Hi, Thanks for the quick replies. I modelled in CME and refined. However, I get a blob of negative density around the S-S bond, which is retained up to 5 sigma. Does it mean it is not CME ? Thanks regards, sreetama On Sunday, 18 January 2015 12:41 AM, Roger Rowlett rrowl...@colgate.edumailto:rrowl...@colgate.edu wrote: If CSO does not account for the density -- the SO bond should be about 1.8 A IIRC -- a possibility is an adventitious metal ion. Roger Rowlett On Jan 17, 2015 1:25 PM, sreetama das somon_...@yahoo.co.inmailto: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
Re: [ccp4bb] additional density on cysteine residue
I suspect it may be a reaction with your reducing agent. What did you use either in the preparation, or in the crystallization. If you didn’t have reducing agent it probably oxidized to sulfuric acid. You should figure it out with difference maps and maybe mass spec also. bob On Jan 17, 2015, at 4:10 PM, sreetama das somon_...@yahoo.co.in wrote: Hi, Thanks for the quick replies. I modelled in CME and refined. However, I get a blob of negative density around the S-S bond, which is retained up to 5 sigma. Does it mean it is not CME ? Thanks regards, sreetama On Sunday, 18 January 2015 12:41 AM, Roger Rowlett rrowl...@colgate.edu wrote: If CSO does not account for the density -- the SO bond should be about 1.8 A IIRC -- a possibility is an adventitious metal ion. Roger Rowlett On Jan 17, 2015 1:25 PM, sreetama das somon_...@yahoo.co.in mailto: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_CME.png all the best! Bob
