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] Se oxidation
Dear all, one comment on the subject of white lines and anomalous phasing power - one has to be extremely careful to jump to conclusions about the effect that a chemical treatment of the heavy atom (say oxydation/reduction of Se) has on the shape of the edge and the associated anomalous signal: there are crystals where the intrinsic anisotropy of the dispersive and anomalous scattering combines with the geometry of the heavy-atom arrangement and the symmetry of the lattice to give a very strong dependency of the edge (and therefore the signal) on the orientation of the sample in the polarized beam. In other words you may observe a wonderful white line but to check if this improved edge comes from oxydising/reducing the sample that went into the crystal you would need to check it by reorienting the crystal in a number of orientations. And of course one would want to do the same on a crystal comign from the protein before the treatment. And I would not be surprised if in some cases the phenomenon would make the difference between succeeding in determining the structure or not - even when non chemical treatment is involved - just a series of crystals of the same kind but mounted and measured in crucially different orientations. See Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments. Schiltz M, Bricogne G. Acta Crystallogr D Biol Crystallogr. 2008 Jul;D64(Pt 7):711-29. Epub 2008 Jun 18. and references cited therein. Regards Pietro -- Pietro Roversi EP Abraham Fellow in Biochemistry - Lincoln College - Oxford Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3RE, England UK Tel. 0044-1865-275385
Re: [ccp4bb] NCS application in Coot, was Re: [ccp4bb] CCP4BB Digest - 8 Feb 2009 to 9 Feb 2009 (#2009-41)
Dear Geoffrey, it seems you read the manual (http://www.ysbl.york.ac.uk/~emsley/coot/doc/chapters/user-manual_5.html#SEC137), but you didnt replace the imol and master-chain-id with a value. You want to specify the molecule (imol, e.g. 0) and the chain from which to copy to others (e.g. A), so: |(copy-from-ncs-master-to-others /imol/ /master-chain-id/)| e.g. scheme/guile: |(copy-from-ncs-master-to-others 0 A)| python: |copy_from_ncs_master_to_others(0, A)| Another way to apply NCS is via Extensions: Extensions-NCS-Copy NCS Chain. Hope this helps, Bernhard P.S. There is a Coot BB: http://www.jiscmail.ac.uk/lists/coot.html Geoffrey Feld wrote: This post has a question and an answer (good karma)...answer first In response to Matt's question about periplasmic harvesting, we do an osmotic lysis of e.coli by a series of centrifugations. First, harvest your cells normally (4k 15 min). Then vigorously resuspend in a 20% (w/v) sucrose, 1 mM EDTA + buffer (we use 20 mM tris). My best yields are 1-2 mL sucrose buffer per 1 mg of protein you expect to get. Equilibrate at RT ~20 min before centrifuging 8k 15 min. Then resuspend the pellets in 5 mM MgSO4 same vol ratio as before (should see foam if you did it right). Finally, spin that down 9k 15 min and collect your periplasmic lysis. You'll want to adjust the pH of the product, depending on what column you want to purify, ours comes out 7 and needs to be 7 for Q sepharose. Enjoy! And my question: I have 3 distinct NCS pairs in the structure I'm refining, and I'd like to make my job a bit easier by applying the NCS edit function in COOT after a round of tinkering. However, when I go to type in the command: copy-from-ncs-master-to-others imol master-chain-id, COOT just sits there like it doesn't know what I'm talking about. Maybe I'm not telling it the proper syntax for master-chain-id? I'm using COOT 0.5.2. 'Preciate it, Geoff -- Geoffrey K. Feld College of Chemistry University of California, Berkeley Vigilia pretium libertatis
Re: [ccp4bb] phaser brute rotation function: how to output .rlist?
Hi, I've just run some tests with Phaser-2.1.4 (the version distributed with CCP4 6.1 and with Phenix-1.3-final), and I get a .rlist file when running the brute rotation function. Could you send me the logfile from your job (probably better to do this off-line) so I can see whether it's possible to explain why you're not getting one? Thanks. Randy Read On 9 Feb 2009, at 23:52, Allyn Schoeffler wrote: Hello all, We've recently installed the newest version of PHASER, and when I do a brute rotation function, I don't get an .rlist file output even though the job completes successfully. Has anyone else had this problem, and is there a way to force PHASER to output an .rlist? (I haven't been able to find the appropriate keyword in the documentation) Thanks very much, Allyn -- Allyn J. Schoeffler Berger Lab Dept. of Molecular and Cell Biology UC Berkeley phone: (510) 643-9491 -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www- structmed.cimr.cam.ac.uk
Re: [ccp4bb] Se oxidation
Dear Savvas, Since you mention the case of TolC explicitly, I should add to what Pietro Roversi has already written in his message in relation to anisotropy effects. Ben Luisi's group kindly let us have their data in the late 20th century, so that we could use the then new and exciting log-likelihood gradient maps in SHARP to take a look at the Se atoms, expecting to see some features around them that would have indicated at least partial oxidation. To our surprise, there was no hint of any such extra density. Later, in papers that paved the way towards the work referred to in Pietro's message, it was pointed out that the anisotropy of anomalous scattering can sometimes result in white lines being visible or not in the Se fluorescence spectra, depending on the orientation of the crystal. The effets can be very strong, and an unlucky orientation for some crystals with a polar axis can be seriously detrimental. See: X-ray absorption, refraction and resonant scattering tensors in selenated protein crystals: implications for data collection strategies in macromolecular crystallography (2005). G. Bricogne, S. C. Capelli, G. Evans, A. Mitschler, P. Pattison, P. Roversi M. Schiltz. J. Appl. Cryst. 38, 168-182. Polarization-dependence of anomalous scattering in brominated DNA and RNA molecules, and importance of crystal orientation in single- and multiple-wavelength anomalous diffraction phasing (2007). R. Sanishvili, C. Besnard, F. Camus, M. Fleurant, P. Pattison, G. Bricogne M. Schiltz. J. Appl. Cryst. 40, 552-558. In section 3.6 of the first paper, a discussion is given of the possibility that what had been interpreted as an oxidation effect (leading to the loss of a white line at the Se K-edge) in selenated N-Myristoyl transferase could plausibly have been an anisotropy (i.e. polarisation dependence) effect. No such re-examination of the TolC case has so far been performed, and now would clearly be a good time to do that. To get back to the initial question: any attempt to interpret in purely chemical terms (e.g. oxidation) what may modify the appearance of the Se K-edge should bear in mind that a physical effect - the polarisation dependence of anomalous scattering - may also be involved in some cases. As shown in the paper referred to in Pietro's message, this was seen as a nuisance and was ignored for the past 20 years, but it can be exploited to extract more phase information from the same datasets; and it is possible to design experiments specifically so as to maximise the amount of extra phase information that this will deliver. With best wishes, Gerard. -- On Tue, Feb 10, 2009 at 08:05:49AM +0100, Savvas Savvides wrote: I think that SeMet oxidation has been a problem in the past in at least one case that I know, that of TolC by Koronakis et al. The same group addressed these problems in more detail in a second paper (see below): Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. Nature. 2000 Jun 22;405(6789):914-9. Oxidation of selenomethionine: some MADness in the method! Sharff AJ, Koronakis E, Luisi B, Koronakis V. Acta Crystallogr D Biol Crystallogr. 2000 Jun;56(Pt 6):785-8. Best wishes Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of aka akaka Sent: Monday, February 09, 2009 7:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Se oxidation Dear All I would like to know whether oxidation of Se entails any problem for SAD or MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents in my protein (extracellular and disulphide bonds are important). Thanks Dr. R.Depetris Weill Cornell Medical College _ Get 5 GB of storage with Windows Live Hotmail. Sign up today. http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_11 2008 E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11720 http://www.pctools.com/en/spyware-doctor-antivirus/ -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * *
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] protocol for harvesting proteins from bacterial periplasmic space
Artem, Artem Evdokimov wrote: Please note that osmotic shock extraction typically employs EDTA which is obviously bad for IMAC. This is not entirely correct. I have used extracts with 5 mM EDTA for IMAC in the past. If your IMAC column volume is large enough, only the top 1-2 mm will be depleted of Me2+ (easily seen with Cu2+). Moreover, it is always possible to add some Me2+ to your extract prior to IMAC. All this was published long ago (Biochemistry. 31: 2690-2702, 1992). Nadir -- Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr
[ccp4bb] Getting a copy of ESCET
I am trying to get a current version of ESCET to compare several models of the same protein with different ligands. I have tried twice to contact Thomas Schneider at the address listed in this post: Hi, for obtaining ESCET, please contact me directly at: thomas.schnei...@embl-hamburg.de Thanks Thomas I have not gotten a reply. I figured the CCP4 bulletin board was my next best shot. Mark
Re: [ccp4bb] ccp4bb] refinment of mir anomalous data in MLPHARE
Hi everyone! I have a problem when I use mlphare Sure you do have a problem: you actually do use mlphare ! ... with no intension to offend Eleanor's favorite program, mlphare was a step forward in phasing, but about 20 years ago. SHARP really put M(S)IR(AS) phasing in a new dimension, and SOLVE and CNS definitely kept in pace. Lately, phaser and bp3 do an excellent job in SAD phasing. Although all programs have strengths and weaknesses, ten years on the road and (auto)SHARP does not stop to amaze me. the latest example being a couple of weeks ago when all else had failed, and embarrassingly enough a friend pushed me back to SHARP. (this will cost me dearly next time in the pub I am afraid) I would likely not have bothered to comment on using SHARP instead of SOLVE or CNS, since much is up to taste and personal experience (... or in other words I personally still prefer SHARP ...) but I am sorry to say that I find playing with mlphare for a MIRAS case to be a waste of time. SHARP would always produce a better map and its so much more flexible to describe your experiment properly ... A. PS1 SHARP is free for academic users and despite older testimonials easy to install these days.
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] protocol for harvesting proteins from bacterial periplasmic space
Sure, it's not always 'disastrously bad' to have EDTA (hence my use of the word 'bad' rather than a more categorical statement. Donuts are bad for me yet I can't stop eating them :) Yes, you can take a risk. However since periplasmic isolation is already a PITA, why add an extra concern? Artem Artem, Artem Evdokimov wrote: Please note that osmotic shock extraction typically employs EDTA which is obviously bad for IMAC. This is not entirely correct. I have used extracts with 5 mM EDTA for IMAC in the past. If your IMAC column volume is large enough, only the top 1-2 mm will be depleted of Me2+ (easily seen with Cu2+). Moreover, it is always possible to add some Me2+ to your extract prior to IMAC. All this was published long ago (Biochemistry. 31: 2690-2702, 1992). Nadir -- Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr
[ccp4bb] bent helix
Dear all, I have a crystal structure of a protein whose first 40 residues are in a helical conformation. This helix was found to be bent, with the bend occurring at residues 27-30, by an angle of roughly 60 deg . We believe this bend was caused by crystal packing forces as this helix nicely packs in with the neighboring symmetry related molecules. I was wondering if anybody has any references of similar cases where conformations have been altered due to crystal packing. Thanks to all in advance. -Shahila Shahila Mehboob, PhD. Visiting Research Assistant Professor Center for Pharmaceutical Biotechnology, M/C870 University of Illinois at Chicago 900 South Ashland Ave. Chicago, IL 60607 Phone: 312-413-9304 (lab) Phone: 773-531-5470 (cell) Email: shah...@uic.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/
