Re: [ccp4bb] unknown density for a small molecule
Dear Mengxia, the lower part could well be a sulfate ion. For the upper part, it is difficult to guess from a static picture. Unfortunately, at the resolution you have there are no programs which can build an unknown small molecule from scratch according to the density. You have to guess, fit it and refine and look if it's ok. I would also check whether a natural ligand of the protein (substrate, product, effector molecule) would fit. They sometimes remain attached during the whole purification procedure. A trick to get an idea where heavy atoms like sulfur are located is to scroll the contour level up. If the density for e.g. the center disappears very late, when most of the other density is no longer visible, it must be a sulfur or other heavy atom. Good luck! Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of mengxiao lv Sent: Tuesday, February 10, 2009 6:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unknown density for a small molecule Dear All, When I was refining my structure, I found some unmodeled blobs, shown as attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor 0.18, refined to 1.9 angstrom ). The protein was expressed in E.coli and purified by nickel column and gel filtration, both in tris buffer. The crystallization condition has 2 M ammonium sulfate and 0.1 M sodium acetate. The lower part looks like a sulfate group, which is held by one Arg, one his, one lys and one asn. The latter three residues are from another asymmetric unit. The other end of the small molecule is stunk by the rings of Tyr and Phe. It also interacts with the OH group of another tyr and one water molecule. Is there a program can build small molecule models according to the densities? Or could anyone tell what it might be from the density? Thanks a lot! Any suggestions will be appreciated! Mengxiao
Re: [ccp4bb] unknown density for a small molecule
Hi, It could be all sorts of things, but the one that for some reason is stuck in my mind is isopentenyl phosphate (phosphate, not pyrophosphate!). Of course w/o seeing the density in 3D this is just a guess. Artem Dear All, When I was refining my structure, I found some unmodeled blobs, shown as attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor 0.18, refined to 1.9 angstrom ). The protein was expressed in E.coli and purified by nickel column and gel filtration, both in tris buffer. The crystallization condition has 2 M ammonium sulfate and 0.1 M sodium acetate. The lower part looks like a sulfate group, which is held by one Arg, one his, one lys and one asn. The latter three residues are from another asymmetric unit. The other end of the small molecule is stunk by the rings of Tyr and Phe. It also interacts with the OH group of another tyr and one water molecule. Is there a program can build small molecule models according to the densities? Or could anyone tell what it might be from the density? Thanks a lot! Any suggestions will be appreciated! Mengxiao
Re: [ccp4bb] unknown density for a small molecule
Hi, If you could send in stereo mode of the density you want to get suggestion, it will be convenient for the well trained eyes. S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany. On Tue, Feb 10, 2009 at 3:16 PM, ar...@xtals.org wrote: Hi, It could be all sorts of things, but the one that for some reason is stuck in my mind is isopentenyl phosphate (phosphate, not pyrophosphate!). Of course w/o seeing the density in 3D this is just a guess. Artem Dear All, When I was refining my structure, I found some unmodeled blobs, shown as attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor 0.18, refined to 1.9 angstrom ). The protein was expressed in E.coli and purified by nickel column and gel filtration, both in tris buffer. The crystallization condition has 2 M ammonium sulfate and 0.1 M sodium acetate. The lower part looks like a sulfate group, which is held by one Arg, one his, one lys and one asn. The latter three residues are from another asymmetric unit. The other end of the small molecule is stunk by the rings of Tyr and Phe. It also interacts with the OH group of another tyr and one water molecule. Is there a program can build small molecule models according to the densities? Or could anyone tell what it might be from the density? Thanks a lot! Any suggestions will be appreciated! Mengxiao
Re: [ccp4bb] unknown density for a small molecule
Hi Mengxiao, The density sure reminds me of a nucleotide. Note the pi-pi stacking of the planar part between Tyr and Phe, and in the third stereo picture you attached, projections at what could be the 2 and 6 positions remind me of Guanine... plus there are Arg, Lys, etc. pointing toward where there could be a phosphate group. Even if the density is not quite 'big enough' to fit it, I'd give it a try. In addition to components of your crystallization conditions, you might consider that something was either co-purified with your protein, or is contaminating your reagents. Good luck! -pamela At 11:37 AM -0500 2/10/09, mengxiao lv wrote: Thank you so much for the replies! I should clarify in my mail that the two images are actually from one blob, just viewed from two sides. Sorry for the confusing. I enclosed some stereo densities for the same blob in this mail. As Artem said, it is very similar to isopentenyl phosphate. The upper part is planar. I agree with Marc and Eleanor's suggestion that the molecule is from the crystallization solution. I have tried to model one acetate into the density, and it fits well. And the lower part can also model into a sulfate. However, I will see the positive density for the missed link between acetate and sulfate. THe pH for crystallization is 4.6. Will acetate form some compound with sulfate? To Jan and Rajesh, I didn't use MES buffer in the whole procedures. Also, the ring in MES might be too large for the upper part. I also get suggestions to model a molecule with a sugar ring, from Kornelius, Poul, Daniele. The images I attached are not clear, and I don't think the density is large enough for a five or six member ring. Thanks again for the help! Hope the new images will make things clear. I appreciate any suggestions! Mengxiao On Tue, Feb 10, 2009 at 2:08 AM, Kontopidis George mailto:gkontopi...@vet.uth.grgkontopi...@vet.uth.gr wrote: Dear Mengxiao, From my experience I would say that the two e. densities (blob1 and blob2) are present the same molecule. What that might be is more difficult to answer. Based in the concentration you gave us and the electron density volume I would say that is more likely to be ammonium sulphate (NH4 and SO4). As you say the lower part looks like SO4 and make sense to interact with Arg, Lys and Asn and the other end looks like NH4. But those two group there and give it one round of refinement. Check the B factors at the end Do they make sense (directions of H-bonds, distance between NH4 and SO4)? Is the Bfactor similar for NH4 and SO4 They should not have a difference greater than 50%. You could try also Na in stead of NH4 George From: CCP4 bulletin board [mailto:mailto:CCP4BB@JISCMAIL.AC.UKccp...@jiscmail.ac.uk] On Behalf Of mengxiao lv Sent: Tuesday, February 10, 2009 7:24 AM To: mailto:CCP4BB@JISCMAIL.AC.UKCCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unknown density for a small molecule Dear All, When I was refining my structure, I found some unmodeled blobs, shown as attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor 0.18, refined to 1.9 angstrom ). The protein was expressed in E.coli and purified by nickel column and gel filtration, both in tris buffer. The crystallization condition has 2 M ammonium sulfate and 0.1 M sodium acetate. The lower part looks like a sulfate group, which is held by one Arg, one his, one lys and one asn. The latter three residues are from another asymmetric unit. The other end of the small molecule is stunk by the rings of Tyr and Phe. It also interacts with the OH group of another tyr and one water molecule. Is there a program can build small molecule models according to the densities? Or could anyone tell what it might be from the density? Thanks a lot! Any suggestions will be appreciated! Mengxiao Content-Type: image/jpeg; name=stereo1.jpg Content-Disposition: attachment; filename=stereo1.jpg X-Attachment-Id: f_fr0sc9ly0 Attachment converted: gerolsteiner:stereo1.jpg (JPEG/«IC») (001FB8B3) Content-Type: image/jpeg; name=stereo2.jpg Content-Disposition: attachment; filename=stereo2.jpg X-Attachment-Id: f_fr0scdg81 Attachment converted: gerolsteiner:stereo2.jpg (JPEG/«IC») (001FB8B4) Content-Type: image/jpeg; name=stereo3.jpg Content-Disposition: attachment; filename=stereo3.jpg X-Attachment-Id: f_fr0sci0r2 Attachment converted: gerolsteiner:stereo3.jpg (JPEG/«IC») (001FB8B5) -- Pamela J. Focia, Ph.D. Research Assistant Professor Structural Biology Facility Manager Robert H. Lurie Comprehensive Cancer Center in the Departments of: Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, and Biochemistry, Molecular Biology Cell Biology, Northwestern University 303 E. Chicago Ave., S-215, Chicago 60611 (312)503-0848 fax (312)503-5349 fo...@northwestern.edu
Re: [ccp4bb] unknown density for a small molecule
It could be purine or pyrimidine ring stacked between the Phe an Tyr. check pdb 3etr or 3eub of xanthine oxidase binding purine substrates between two Phe at the active site. Hongnan Cao UCR Date: Tue, 10 Feb 2009 00:23:54 -0500From: lvmengx...@gmail.comsubject: [ccp4bb] unknown density for a small moleculeTo: ccp...@jiscmail.ac.ukdear All,When I was refining my structure, I found some unmodeled blobs, shown as attached images (contoured at 3 sigma for Fo-Fc, Rfree 0.21 and Rfactor 0.18, refined to 1.9 angstrom ). The protein was expressed in E.coli and purified by nickel column and gel filtration, both in tris buffer. The crystallization condition has 2 M ammonium sulfate and 0.1 M sodium acetate. The lower part looks like a sulfate group, which is held by one Arg, one his, one lys and one asn. The latter three residues are from another asymmetric unit. The other end of the small molecule is stunk by the rings of Tyr and Phe. It also interacts with the OH group of another tyr and one water molecule. Is there a program can build small molecule models according to the densities? Or could anyone tell what it might be from the density? Thanks a lot! Any suggestions will be appreciated!Mengxiao _ 上Windows Live 中国首页，下载最新版 MSN！ http://im.live.cn/
