Dear Kavyashree, It is possible that your bound ligand (for which you have strong electron density) is actually a break-down of the parent compound? We have seen this a couple of times now.
Also - are the poorly refining areas (those with negative density) part of a pendant ring connected by a conformationally unrestricted bond? These quite often have poor density. Hard to judge without seeing the actual density - but understand why! Regards, Antony. Sent from my iPhone On 25 May 2013, at 10:40, "Kavyashree Manjunath" <[email protected]> wrote: > Dear Sir, > > Thank you all for your kind advice and clarifications. > I will keep the occupancy 1.0 for both the ligands and > refine without considering the negative density in this > case. > > Thanking you > Regards > Kavya > > > >> -----BEGIN PGP SIGNED MESSAGE----- >> Hash: SHA1 >> >> Dear Kavya, >> >> I don't see much sense in having different occupancies within the same >> molecule (unless one atoms sits on a special position, but then refmac >> will take care of it). >> >> If positive density comes up it's a good sign the ligand really is >> there. At 2.2A I would not be too surprised some atoms show less >> density than others (but don't look too much at the map with a sigma >> level < 1: you are going to see what you want to see). >> >> It's difficult to judge without sitting next to you, so my advice is >> try to model it as good as your knowledge permits, and do take your >> chemical knowledge into account when doing so! >> >> Best, >> Tim >> >> On 05/25/2013 06:55 AM, Kavyashree Manjunath wrote: >>> Dear users, >>> >>> I tried giving occupancy of 0.65 and 0.6 respectively for all atoms >>> of the two ligands and refined. Now after refinement, the atoms of >>> ligand does not have negative density but those which did not have >>> negative density previously appear positive. So what do I need to >>> do? under what circumstances does a ligand have different >>> occupancies for different atoms or for a group of atoms. Any such >>> references are very much welcome. >>> >>> Thank you Regards Kavya >>> >>>> Sir, >>>> >>>> Yes it is around ligand. The average B-factor of one of the >>>> ligand is 36.78, of which one of the atom has occupancy B >>>> factor 0.58 39.37 0.56 38.77 0.87 >>>> 37.00 Three atoms are of same type. The other ligand's overall >>>> Bfactor is 17.64. occupancy B factor 1.00 19.29 >>>> 0.64 23.90 Two atoms are of same type. >>>> >>>> So in the present case should I put the occupancy of 0.56 for all >>>> atoms of ligand-1 and 0.64 for all atoms of ligand-2 and refine? >>>> >>>> I mean will it be wrong to put different occupancies for >>>> different atoms of same ligand? >>>> >>>> I could not see any alternate densities coming up for those >>>> atoms which did not have densities. But 2FoFc map would appear >>>> around these atoms at 0.7 sigma at the same position where the >>>> atoms are present. >>>> >>>> Thank you Regards Kavya >>>> >>>>> Hi Kavya, >>>>> >>>>> If I understand you correctly (from title and text), you meant >>>>> your negative FoFc was around your ligand, is that right? I >>>>> wonder if this is a case in which the ligand has an occupancy >>>>> below 1, but was modeled as 1, so the refinement program had to >>>>> give it a high B factor to compensate that, which results in >>>>> the electron density bleeding into nearby space where there >>>>> should not be so much of it. If you want to test this >>>>> hypothesis, one thing you can try is to set the occupancy to >>>>> 0.25, 0.5,0.75 and so on, and refine a few cycles to see what >>>>> happens to the maps. Also, what's the B factor of the ligand, >>>>> and what's the B of the nearby protein atoms? The difference >>>>> between them can also give you some hint for guessing the >>>>> ligand's occupancy. Some refinement programs(phenix.refine and >>>>> shelx) can also let you refine the ligand's occupancy. >>>>> >>>>> As to the "missing" atoms, that could be caused by alternative >>>>> conformations of the ligand - assuming you have already done a >>>>> thorough refinement. >>>>> >>>>> Zhijie >>>>> >>>>> -------------------------------------------------- From: >>>>> "Kavyashree Manjunath" <[email protected]> Sent: Friday, >>>>> May 24, 2013 12:50 PM To: <[email protected]> Subject: >>>>> [ccp4bb] Negative FoFc around ligand >>>>> >>>>>> Dear users, >>>>>> >>>>>> I am using refmac 5.7.0029 for refining a structure >>>>>> (resolution 2.2 Ang) bound to 2 ligands. After MR There is a >>>>>> very clear density of ligands but after refinement, I get >>>>>> negative fofc map near one of the ligand upto 5 sigma. >>>>>> However its 2fofc map covers the whole ligand. Also for the >>>>>> other ligand, I do not see any 2fofc density (at 3 sigma) for >>>>>> 2 atoms, without these atoms the ligand is unrealistic. But >>>>>> the density comes up around these at around 0.7 sigma. >>>>>> Overall completeness is 99.9% Rmerge 7.5% >>>>>> >>>>>> What else I need to check in the data. Kindly provide some >>>>>> suggestions. >>>>>> >>>>>> Thanking you Regards Kavya >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> -- This message has been scanned for viruses and dangerous >>>>>> content by MailScanner, and is believed to be clean. >>>>> >>>>> >>>>> -- This message has been scanned for viruses and dangerous >>>>> content by MailScanner, and is believed to be clean. >>>> >>>> >>>> >>>> -- This message has been scanned for viruses and dangerous >>>> content by MailScanner, and is believed to be clean. >> >> - -- >> Dr Tim Gruene >> Institut fuer anorganische Chemie >> Tammannstr. 4 >> D-37077 Goettingen >> >> GPG Key ID = A46BEE1A >> -----BEGIN PGP SIGNATURE----- >> Version: GnuPG v1.4.12 (GNU/Linux) >> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ >> >> iD8DBQFRoHp0UxlJ7aRr7hoRAti/AJ9PQQE8cgccPFuaWN842qlwkCK8FgCg/BPR >> 8485l18SMJ4Th5XD0X5be1w= >> =GfVo >> -----END PGP SIGNATURE----- >> >> -- >> This message has been scanned for viruses and >> dangerous content by MailScanner, and is >> believed to be clean. > > > > -- > This message has been scanned for viruses and > dangerous content by MailScanner, and is > believed to be clean.
