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 <[email protected]> (on behalf of Mark J van >Raaij <[email protected]>) >Subject: Re: [ccp4bb] Enigmatic electron density attached to Cys residue >To: [email protected] > >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: >> [email protected] >> >> Homepage: >> http://www.bcp.fu-berlin.de/chemie/bc/ag/agwahl/ >> <coot1.png><coot2.png><coot3.png>
