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What you're seeing is not necessarily pyrophosphate. The negative difference density appears to concentrate around oxygen atoms most distant from the protein (and perhaps around the putative phosphorous atom?). Some clues might be obtained from e.g. the B-factor profile of the refined atoms. From the side-view pictures you sent it's not clear whether the density in question suports two tetrahedral ions... instead it could be two planar ions e.g. a malonate or some other molecule with two flat groups and a linker. Take a closer look at your omit difference density maps for starters. Does the positive difference density indicate the presence of a heavier atom in the center of a putative tetrahedron of lighter ones? Is one of the sides tetrahedral and the other - planar? Incidentally, if you compress the images you sent it's possible to get the size down from 3 MB each (encoded for email it's actually 4MB each!) down to about 60-100 KB each without significant loss of quality. I am sorry for bringing up my pet peeve but not everyone on this list enjoys 8 MB mass emails, especially when the contents do not justify the size in any way :) Artem -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Junyu Xiao Sent: Sunday, July 19, 2009 7:30 PM To: [log in to unmask] Subject: [ccp4bb] Density for pyrophosphate? Dear all, I am at the late stage of refining a 1.97 A structure. There is something in the ligand binding pocket, and I am not sure what it is exactly. Since this protein can potentially generate pyrophosphate from ATP, and I have ATP in the protein solution, so I tried to fit in a pyrophosphate. The Rwork/Rfree after refinement are 0.198/0.243. Below is the refined map. The 2fofc map looks ok (contoured at 1 sigma), however, there is always some red density around the pyrophosphate in the fofc map (contoured at 3 sigma). I have also tried to fit in the density as two free phosphates, and the results are worse. Can I get some advices on this? Any suggestion will be greatly appreciated. Best regards, Junyu