Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement
Thanks James and all of you, The occupancy had to be lowered to 0.25 to get the B-factor similar to the coordinating atoms. But now I can see a good positive density. Ivan On Tue, Jun 29, 2010 at 7:49 PM, James Holton jmhol...@lbl.gov wrote: This kind of thing happens when the metal is not at 100% occupancy. Or if it not what you think it is (Mg instead of Ca, for example). Depending on your refinement program, you can do occupancy refinement or a poor man's occupancy refinement where you adjust the occupancy manually and then refine the B factors. When your unknown atom has a B factor similar to its neighbors, you are probably at about the right occupancy. -James On 6/29/2010 6:35 PM, xaravich ivan wrote: Dear CCP4BB, I have come across something that might be pretty obvious to experienced people but is making me crazy. I have this great 1.15 angs data and I know that I have a Calcium ion (pics attached) from previous structures of the same protein, that I have solved. Rightly when I add waters with Arp solvent it does not put water at that positive density. Now whenever I have tried to put the calcium, and refine the structure it is giving me a negative density at the metal site. I csn see that the 2fc-fo is clear there, but why negative density. This is just the start of my refinement and I have to refine multiple ligands in the structure and I would like to get past this issue before that. I tried putting atom at the pointer, adding water and renaming it according to the naming convention in the PDB for Calcium, but nothing. Your suggestions would be invaluable, as always. Ivan
Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement
Negative (or positive) density tells you what you that what you've modelled is wrong. If you have 1.15A X-ray data and it's telling you what you think is Ca2+ is not Ca2+, then I would think your X-ray data wins and your previous structures lose (a lot can happen from one structure to the next...). I must say, your 2fo-fc looks like a perfectly good Mg2+, since it is the same size (ish) as the surrounding O and C atoms; a Ca2+ would have given a much larger green blob. phx. On 30/06/2010 02:35, xaravich ivan wrote: Dear CCP4BB, I have come across something that might be pretty obvious to experienced people but is making me crazy. I have this great 1.15 angs data and I know that I have a Calcium ion (pics attached) from previous structures of the same protein, that I have solved. Rightly when I add waters with Arp solvent it does not put water at that positive density. Now whenever I have tried to put the calcium, and refine the structure it is giving me a negative density at the metal site. I csn see that the 2fc-fo is clear there, but why negative density. This is just the start of my refinement and I have to refine multiple ligands in the structure and I would like to get past this issue before that. I tried putting atom at the pointer, adding water and renaming it according to the naming convention in the PDB for Calcium, but nothing. Your suggestions would be invaluable, as always. Ivan
Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement
Hi Ivan, these Dale Tronrud's slides might help you with understanding the maps: http://www.ccp4.ac.uk/courses/stwk10/talk_files/Dale_The_Wonderful_World_of_Maps.pdf Good luck! Pavel. On 6/29/10 6:35 PM, xaravich ivan wrote: Dear CCP4BB, I have come across something that might be pretty obvious to experienced people but is making me crazy. I have this great 1.15 angs data and I know that I have a Calcium ion (pics attached) from previous structures of the same protein, that I have solved. Rightly when I add waters with Arp solvent it does not put water at that positive density. Now whenever I have tried to put the calcium, and refine the structure it is giving me a negative density at the metal site. I csn see that the 2fc-fo is clear there, but why negative density. This is just the start of my refinement and I have to refine multiple ligands in the structure and I would like to get past this issue before that. I tried putting atom at the pointer, adding water and renaming it according to the naming convention in the PDB for Calcium, but nothing. Your suggestions would be invaluable, as always. Ivan
Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement
Could it be that for some reason (like the components of your solutions) you have Mg2+ in that site? Also, Magnesium is a common contaminant, trace amounts are usually present in one chemical or another. Subtle differences in the site might make it a better site for another lighter ion (we still do not fully understand the subtility of protein structures, and of their modifications). Fred. Fred. Message du 30/06/10 03:36 De : xaravich ivan A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : [ccp4bb] Density changes from Positive to negative after ligand addition and refinement Dear CCP4BB, I have come across something that might be pretty obvious to experienced people but is making me crazy. I have this great 1.15 angs data and I know that I have a Calcium ion (pics attached) from previous structures of the same protein, that I have solved. Rightly when I add waters with Arp solvent it does not put water at that positive density. Now whenever I have tried to put the calcium, and refine the structure it is giving me a negative density at the metal site. I csn see that the 2fc-fo is clear there, but why negative density. This is just the start of my refinement and I have to refine multiple ligands in the structure and I would like to get past this issue before that. I tried putting atom at the pointer, adding water and renaming it according to the naming convention in the PDB for Calcium, but nothing. Your suggestions would be invaluable, as always. Ivan [ Screen shot 2010-06-29 at 6.15.29 PM.png (375.3 Ko) ] [ Screen shot 2010-06-29 at 6.21.22 PM.png (383.5 Ko) ]
