[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
Dear Priyank, At 4.5 sigma, it is definitively something heavy like a gold ion. The red sphere of difference density around the ion is most likely caused by the fact that the ion has partial occupancy, which was not refined. In these cases, refinement programs try to compensate for partial occupancy by increasing the Bfactor, resulting in an expansion of the model “electron density”. I would try to refine the occupancy of the ion. If this does not work, you could also manually fix the occupancy at 0.9, 0.8, 0.7 etc. and see at which value the difference density disappears. However, judging from the map the resolution should be sufficient for occupancy refinement of the ion. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von PriyankMaindola Gesendet: Dienstag, 10. Dezember 2013 17:13 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain dear all: I. i am not able to fit trp, since 1. trp doesnt fit well 2. positive density comes in fo-fc map after refinement 3. this is a soaked crystal str with heavy atom soln, the native one has perfect density for gln, so mutation to trp is unlikely II. on increasing contour level, 2fo-fc map fades above 4.5 rmsd if I do not put anything in the blob and see the refined map III. placing Au+ and refining (occ 1 ; B-fac 63 A2) gives figure 4.png (attached below) however anomalous diff map does give positive density but not a clear round, spherical one. On 10 December 2013 19:29, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: My first guess was also a metal ion. However, a tryptophan as Fred suggested cannot be ruled out. A simple preliminary test is to scroll up the contouring level and look when the contours of the blob disappear. If the contours quickly disappear, you have something disordered or light. If the contours of the blob disappear at the same moment or later as e.g. sulfur atoms, you have something heavy like a metal ion. You still have to fit all possibilities and see what refines best. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Matthias Zebisch Gesendet: Dienstag, 10. Dezember 2013 14:21 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain check an anomalous map! The obvious thing to do to rule out gold binding - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.ukmailto:matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 12/10/2013 12:44 PM, PriyankMaindola wrote: dear members i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. please suggest following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd -- Priyank -- Priyank Maindola
[ccp4bb] AW: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
My first guess was also a metal ion. However, a tryptophan as Fred suggested cannot be ruled out. A simple preliminary test is to scroll up the contouring level and look when the contours of the blob disappear. If the contours quickly disappear, you have something disordered or light. If the contours of the blob disappear at the same moment or later as e.g. sulfur atoms, you have something heavy like a metal ion. You still have to fit all possibilities and see what refines best. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Matthias Zebisch Gesendet: Dienstag, 10. Dezember 2013 14:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain check an anomalous map! The obvious thing to do to rule out gold binding - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.ukmailto:matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 12/10/2013 12:44 PM, PriyankMaindola wrote: dear members i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. please suggest following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd -- Priyank
Re: [ccp4bb] AW: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
It doesn't look like you left a CN on your gold atom. These things are pretty much covalently bound. Dale Tronrud On 12/10/2013 08:13 AM, PriyankMaindola wrote: dear all: I. i am not able to fit trp, since 1. trp doesnt fit well 2. positive density comes in fo-fc map after refinement 3. this is a soaked crystal str with heavy atom soln, the native one has perfect density for gln, so mutation to trp is unlikely II. on increasing contour level, 2fo-fc map fades above 4.5 rmsd if I do not put anything in the blob and see the refined map III. placing Au+ and refining (occ 1 ; B-fac 63 A2) gives figure 4.png (attached below) however anomalous diff map does give positive density but not a clear round, spherical one. On 10 December 2013 19:29, herman.schreu...@sanofi.com mailto:herman.schreu...@sanofi.com wrote: My first guess was also a metal ion. However, a tryptophan as Fred suggested cannot be ruled out. A simple preliminary test is to scroll up the contouring level and look when the contours of the blob disappear. If the contours quickly disappear, you have something disordered or light. If the contours of the blob disappear at the same moment or later as e.g. sulfur atoms, you have something heavy like a metal ion. You still have to fit all possibilities and see what refines best. __ __ Best, Herman __ __ *Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Matthias Zebisch *Gesendet:* Dienstag, 10. Dezember 2013 14:21 *An:* CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK *Betreff:* Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain __ __ check an anomalous map! The obvious thing to do to rule out gold binding - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK __ __ Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk mailto:matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 12/10/2013 12:44 PM, PriyankMaindola wrote: dear members __ __ i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. __ __ please suggest __ __ following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) __ __ k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd __ __ __ __ -- *Priyank* __ __ -- *Priyank Maindola*
