Re: [ccp4bb] Enigmatic electron density attached to Cys residue
What odds to you give us? ...I bet on disordered DTT covalently bound to the protein. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 19 Aug 2014, at 16:12, Bernhard Loll wrote: Dear all, We are currently working on a small GTPase. The structure has been solved to 1.4 A with two molecules in the ASU. In the difference electron density we can clearly see difference density (in one monomer) attached to a Cys residue. The protein has been expressed in E. coli. For crystallization experiments the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM HEPES pH 7.5 and 50 mM NaCl. Prior to crystallization the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol. The protein crystallized under the following conditions: 28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0 The anomalous signal is too weak to judge the positions of the sulphur atoms. We have performed MS analysis on the protein before crystallization and on dissolved protein crystals. MS revealed a mass difference of about 135 Da, indicating that some chemistry must have went on in the crystallization drop. The extra electron density has a planar shape and is quite symmetric. We have placed some dummy water molecules in the density. Distances are given in A in the PNG file. Attached files coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron density map (blue) @ sigma=+3 after phenix.refine coot2.png: same electron densities as in coot1.png, with dummy atoms placed coot3.png: same electron densities as in coot1.png, side view Thanks for your time and efforts. Cheers, Bernhard -- Dr. Bernhard Loll Freie Universitaet Berlin Fachbereich Biologie, Chemie, Pharmazie Institut fuer Chemie und Biochemie AG Strukturbiochemie Takustr. 6 D-14195 Berlin Germany Phone: +49 (0) 30 838-57348 Fax: +49 (0) 30 838-457348 Email: l...@chemie.fu-berlin.de Homepage: http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/ coot1.pngcoot2.pngcoot3.png
Re: [ccp4bb] Enigmatic electron density attached to Cys residue
Hi, I guessed glycerol by looking at the figs and not reading your text. Having read your text afterwards, you do have glycerol in the solution. My guess is glycerol. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org Original message Date: Tue, 19 Aug 2014 16:39:36 +0200 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Mark J van Raaij mjvanra...@cnb.csic.es) Subject: Re: [ccp4bb] Enigmatic electron density attached to Cys residue To: CCP4BB@JISCMAIL.AC.UK What odds to you give us? ...I bet on disordered DTT covalently bound to the protein. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 19 Aug 2014, at 16:12, Bernhard Loll wrote: Dear all, We are currently working on a small GTPase. The structure has been solved to 1.4 A with two molecules in the ASU. In the difference electron density we can clearly see difference density (in one monomer) attached to a Cys residue. The protein has been expressed in E. coli. For crystallization experiments the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM HEPES pH 7.5 and 50 mM NaCl. Prior to crystallization the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol. The protein crystallized under the following conditions: 28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0 The anomalous signal is too weak to judge the positions of the sulphur atoms. We have performed MS analysis on the protein before crystallization and on dissolved protein crystals. MS revealed a mass difference of about 135 Da, indicating that some chemistry must have went on in the crystallization drop. The extra electron density has a planar shape and is quite symmetric. We have placed some dummy water molecules in the density. Distances are given in A in the PNG file. Attached files coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron density map (blue) @ sigma=+3 after phenix.refine coot2.png: same electron densities as in coot1.png, with dummy atoms placed coot3.png: same electron densities as in coot1.png, side view Thanks for your time and efforts. Cheers, Bernhard -- Dr. Bernhard Loll Freie Universitaet Berlin Fachbereich Biologie, Chemie, Pharmazie Institut fuer Chemie und Biochemie AG Strukturbiochemie Takustr. 6 D-14195 Berlin Germany Phone: +49 (0) 30 838-57348 Fax: +49 (0) 30 838-457348 Email: l...@chemie.fu-berlin.de Homepage: http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/ coot1.pngcoot2.pngcoot3.png
Re: [ccp4bb] Enigmatic electron density attached to Cys residue
Hi Bernhard, It is difficult guess with two dimensional images. Is it possible a metal coordinated by Cys-Sulfur and one or two acetate ions? HTH, Partha Sent from my iPhone On Aug 19, 2014, at 10:12 AM, Bernhard Loll l...@chemie.fu-berlin.de wrote: Dear all, We are currently working on a small GTPase. The structure has been solved to 1.4 A with two molecules in the ASU. In the difference electron density we can clearly see difference density (in one monomer) attached to a Cys residue. The protein has been expressed in E. coli. For crystallization experiments the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM HEPES pH 7.5 and 50 mM NaCl. Prior to crystallization the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol. The protein crystallized under the following conditions: 28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0 The anomalous signal is too weak to judge the positions of the sulphur atoms. We have performed MS analysis on the protein before crystallization and on dissolved protein crystals. MS revealed a mass difference of about 135 Da, indicating that some chemistry must have went on in the crystallization drop. The extra electron density has a planar shape and is quite symmetric. We have placed some dummy water molecules in the density. Distances are given in A in the PNG file. Attached files coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron density map (blue) @ sigma=+3 after phenix.refine coot2.png: same electron densities as in coot1.png, with dummy atoms placed coot3.png: same electron densities as in coot1.png, side view Thanks for your time and efforts. Cheers, Bernhard -- Dr. Bernhard Loll Freie Universitaet Berlin Fachbereich Biologie, Chemie, Pharmazie Institut fuer Chemie und Biochemie AG Strukturbiochemie Takustr. 6 D-14195 Berlin Germany Phone: +49 (0) 30 838-57348 Fax: +49 (0) 30 838-457348 Email: l...@chemie.fu-berlin.de Homepage: http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/ coot1.png coot2.png coot3.png
Re: [ccp4bb] Enigmatic electron density attached to Cys residue
The planarity and symmetry rules out a mixed disulfide with DTT. Wrong size/shape for glycerol. Without looking at your crystallization reagents, I would have suggested something like an S-hydroxycysteine adduct of something like 2,4-pentanedione or a dehydrated MPD alkene. Could pentanedione be a degradation product of PEG200? Did any MPD get into the mix? We have seen spurious oxidation of Cys to Cys-OH in thiol proteins, e,g, PDB 3UAO. Good luck. With that high quality ED, you'd think it would be easy to ID... Roger Rowlett Dear all, We are currently working on a small GTPase. The structure has been solved to 1.4 A with two molecules in the ASU. In the difference electron density we can clearly see difference density (in one monomer) attached to a Cys residue. The protein has been expressed in E. coli. For crystallization experiments the GTPase was incubated with GMPPNP, that had been dissolved in 20 mM HEPES pH 7.5 and 50 mM NaCl. Prior to crystallization the protein was stored in a buffer composed of 20 mM HEPES pH 7.5, 200 mM NaCl, 5 mM, Mg acetate, 2 mM DTT and 2% (v/v) glycerol. The protein crystallized under the following conditions: 28% (v/v) PEG 200, 5% (w/v) PEG 3000 and 100 mM MES buffer at pH 6.0 The anomalous signal is too weak to judge the positions of the sulphur atoms. We have performed MS analysis on the protein before crystallization and on dissolved protein crystals. MS revealed a mass difference of about 135 Da, indicating that some chemistry must have went on in the crystallization drop. The extra electron density has a planar shape and is quite symmetric. We have placed some dummy water molecules in the density. Distances are given in A in the PNG file. Attached files coot1.png: 2FoFc electron density map (blue) @ sigma=1 and FoFc electron density map (blue) @ sigma=+3 after phenix.refine coot2.png: same electron densities as in coot1.png, with dummy atoms placed coot3.png: same electron densities as in coot1.png, side view Thanks for your time and efforts. Cheers, Bernhard -- Dr. Bernhard Loll Freie Universitaet Berlin Fachbereich Biologie, Chemie, Pharmazie Institut fuer Chemie und Biochemie AG Strukturbiochemie Takustr. 6 D-14195 Berlin Germany Phone: +49 (0) 30 838-57348 Fax: +49 (0) 30 838-457348 Email: l...@chemie.fu-berlin.de Homepage: http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/
