[ccp4bb] Fwd: [ccp4bb] tricky mr problem
I would try phase improvement, especially since you have two molecules per a.u. In other words averaging, solvent flattning, histogram matching. The best way is to start at low(er) resoltuion and extend to the highest resolution vailable. The best criterium for success is a improved and interpretable map. I had quite some success with this appraoch with MR solutions, but uninterpretable or difficult to build maps. Good luck, Klaus Dr. Klaus Piontek Albert-Ludwigs University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de --- the forwarded message follows --- ---BeginMessage--- Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Cheers, Rhys---End Message---
Re: [ccp4bb] Question about TEV cleavage
And not at full moon! Klaus Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu: Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] geometry problems with sugars
Dear sugar loving people, I have been working with a lot of glycoproteins (up to 30 % carbohydrates) at resolutions as bad as 2.8 Å. Nevertheless, I was able to built sometimes about 10 sugar moieties/carbohydrate chain. Although, sugar molecules usually have a somewhat bulky density, and don´t have such distinct structural features like in proteins (e.g. main chain, side chain, C=O bump) one can use geometric restraints to place the sugars correctly with a high probability. I other words I usually first superimpose the e.g. O1 of the sugar molecule with the N of the Asn or O of the Ser/Thr or with the e.g. O4 of the previous sugar (looking from Asn/Ser/Thr). Then I just rotate about the glycosidic bond or only about the O1 (e.g. C1-O4 for a beta1-4) and try to fit the density as good as possible. Often real space refinement in COOT helps after this stage to optimize. Then I check the interactions of the newly placed sugar with its close neighbourhood. Well defined sugars, and only those you can see in a crystal structure, make practically with each of their OH groups or any other polar atom/group (e.g. the N in NAG) at least one H-bond, either with the protein, with other neighbouring sugars, or with waters or even with other solvent molecules like SO4. Then you should also check reasonable van der Waals distances of non-polar atoms. If you find a conformation where you have the optimum number of H-bonds and no outliers (e. g. 3.2 Å) of close non-bonding contacts, you most likely have placed your sugar molecule correctly. In e.g. REFMAC with the review mode you can then check if you have a alpha or beta glycosidic bond and if this is what you expect. BR, Klaus Am 22.04.2010 um 19:13 schrieb tirumal: Thanks to all who responded. 180 degrees flip of the problematic NAGs, did help. At the moment, there is no substitute for knowledge when building carbohydrates - it would be a substantial improvement I think if someone added intelligent carbohydrate validation tools into Coot. If you have a poor density (which I guess, generally is the case for large glycoprotein structures) you have to depend on trial and error strategy to get the right NAG conformation. I don't know how other refinement programs handle this, but after Phenix.refinement run, one has to definitely check the geometry of the NAGs carefully. Hope to see a validation tool for NAGs in Coot soon. Tirumal --- On Wed, 21/4/10, Garib Murshudov ga...@ysbl.york.ac.uk wrote: From: Garib Murshudov ga...@ysbl.york.ac.uk Subject: Re: [ccp4bb] geometry problems with sugars To: CCP4BB@JISCMAIL.AC.UK Date: Wednesday, 21 April, 2010, 9:58 JED's example is very illustrative and it shows that chirality may need to be added to this link definition. then sugar validation may be easier (at least ASN-NAG with only one sugar). If chirality is wrong then rotate around ND2-C1bond as a rigid group. Just like you do with rotamers. Here you have only two orientations. Garib On 21 Apr 2010, at 14:20, Paul Emsley wrote: Garib Murshudov wrote: As I see there is no chirality definition for NAG-ASN link (perhaps there should be but then people will be unhappy even more). Only reason i can see for this flattening is conflict between geometry and electron density. Your example shows that even if electron density is weak it may play a role and correct orientation of sugar may matter. I agree, and with JED too. More tests suggest that if I put the NAG into the density the wrong way round, Coot will happily flatten the C1. So, my guess would be that if you rotated your NAG 180 degrees round a vector ~ NG--(midpoint of C3,C4) and re-refined, then things would improve. At the moment, there is no substitute for knowledge when building carbohydrates - it would be a substantial improvement I think if someone added intelligent carbohydrate validation tools into Coot. Paul. Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
[ccp4bb] publBio
Dear Xtalographers, has someone out there ever used publBio, a tool for preparing manuscripts for publication (e.g. to Acta Cryst.)? What was your experience? BR, Klaus Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] Molecular replacement using a partial molecular replacement solution
Wong, did you look for pseudo-symmetry? For example for pseudo-translation, which you can deduce from a native Patterson. Good luck, Klaus Mo Wong wrote: Hello, My problem: I have poorly phased 3.5A data which suggests 6 molecules per ASU, and using MolRep with the experimental phases (search for model in the map) I have good solutions 3 of them. There is a lot of empty electron density which needs to be filled with more copies of the molecule. I have looked for NCS operators as I know this will improve the map and help with model fitting (the Self Rot function suggests I have at least 2-fold symmetry), but no luck yet. My current focus is on using the 3-molecule partial solution as a starting point, but since I'm not getting anywhere fast, I though I'd post to the bulletin board. Can someone please either point me to a MolRep script that allows me to fix the known solutions, use the experimental phases, and search for (3?) more copies of the model, or tell if there is something wrong with the following Phaser scripts below (is it necessary to apply an operator to the MolRep solution before reading into Phaser?). Thanks! # phaser eof MODE MR_FRF HKLIN overall_best_denmod_map_coeffs.mtz LABIN F = FP SIGF = SIGFP ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25 ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25 COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1 COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3 SOLU 6DIM ENSE trimer EULER 0 0 0 FRAC 0 0 0 SEARCH ENSEMBLE monomer NUM 1 ROOT AUTO_monomer eof # phaser eof MODE MR_FTF HKLIN overall_best_denmod_map_coeffs.mtz LABIN F = FP SIGF = SIGFP ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25 ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25 COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1 COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3 @AUTO_monomer.rlist ROOT AUTO_monomer2 eof # -- Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] tricoordinated ion?
Hi Sebastiano, I had similar experiences with various crystal structures. After a lot of modeling (albeit at about 1 A resolution) I came to the conclusion that these atoms correspond to disordered solvent molecules, e.g. water and/or Zn. In other words the atoms have occupancies of 1, but in the sum it should be 1. For CO2 or similar molecules the bond length are far too long (C-O is about 1.4 A or so). Good luck. Klaus Piontek Sebastiano Pasqualato wrote: Hi all, I wanted to ask you what would you model in the density in which I have at the moment modelled 4 water molecules, which are however too close to be waters, I guess (see attached image). My crystallisation conditions contain NaCl, MgCl2, Peg400, TrisHCl, TCEP, glycerol. I can't think at a tricoordinated ion like that... thanks in advance for the hints, ciao S -- Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: [EMAIL PROTECTED] Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
[ccp4bb] COOT H-atoms
Dear COOT users, does anybody know about a possibility to move single hydrogen atoms or groups of it (e.g. -CH3) during model building in COOT? Either by rotation about a dihedral angle or just by a translational movement. Any advice in this direction is highly appreciated. Regards, Klaus -- Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: [EMAIL PROTECTED] Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] Arp/warp space group P 21 2 21
Probably because it's not a standard notation. The standard notation is P21212. I would reindex and run again. Klaus PhilEvans wrote: Is there a reason why Arp/warp doesn't like space group P 21 2 21? Phil -- Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: [EMAIL PROTECTED] Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] Oxidized manganese?
Dear Tiancen, I would look to the coordination of the Mn. In other words to the geometry and distances from the Mn to it's potential ligands. The most stable oxidation state of Mn is +2, with a octahedral (6-fold) coordination sphere and bond lengths (e.g. Mn-O) of 2.1-2.4 Å. For Mn3+ one would expect somewhat longer bonds. But Mn3+ is probably not stable in the environment of a protein. Even if the Mn2+ was oxidized and stayed like that, during data collection (at least at a high dose like at a synchrotron) one could expect reduction through X-ray radiation. Greetings, Klaus TC Hu wrote: Dear all, Sorry for the non-topic question. We are working on an enzyme with two Mn2+ in its active center. The Mn2+-coordinated water molecules are responsible for binding and nucleophillic attack of the substrate. We soaked the protein crystal with an inhibitor and determined the structure, in which we did not find the inhibitor. However, from the Fsoaked_crystal - Funsoaked_crystal map constructed with the refined phases, we discovered two strong (4 sigma) difference signal near the two Mn2+, one for each. Both two datasets were collected to 2.2A and of good quality. Considering the strong effect of the inhibitor (IC50 ~75nM), we doubt that the manganese ions might be oxidized and the catalytic water molecules are replaced. But since there are several oxidation states of manganese, how can we verify which one occurred in our crystal? Another minor question: is there any movie-making software that can produce the animation of bond formation and breakage (like that during catalysis)? Thanks for your input! Tiancen Hu Shanghai Institute of Materia Medica Chinese Academy of Sciences -- Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: [EMAIL PROTECTED] Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
[ccp4bb] Postdoc and PhD position
*A post-doc position and a PhD position in structural biology is available in the group of * *Prof. Dietmar A. Plattner* *at* *Institute of Organic Chemistry and Biochemistry, Albert-Ludwigs-University Freiburg/Germany* The project involves investigations of structure-function relationships of fungal ligninolytic redox enzymes (e. g. peroxidases, laccases, ref. Blodig et al., JMB, 851, 2001; Piontek et al., JBC, 37663, 2002) by means of X-ray crystallography. The studies will be part of the integrated project BIORENEW (6th EU-Framework Programme), which involves 26 partners from academic institutions and from industry. Further information under www.chemie.uni-freiburg.de/orgbio/w3platt *Requirements:* I) Post-doc position: PhD in protein crystallography. Experience with purification and crystallization of proteins, data collection and processing, structure determination, refinement, modeling and analysis of model structures. Knowledge of enzyme kinetic and UNIX is a plus. II) PhD position: Graduation in chemistry or a related field, preferably with a biochemical topic. Experience with purification, crystallization or X-ray structure determination of proteins would be an asset. Both positions: The ability to work independently and team spirit. The positions are open immediately. Employment will be according to the regulations of the German public service (50 % for the PhD position, no tuitions fees). *Application:* Send your CV and a letter of recommendation by email to: Dr. Klaus Piontek Email: [EMAIL PROTECTED] Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 -- Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: [EMAIL PROTECTED] Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
[ccp4bb] Fwd: [ccp4bb] Swiss humour - no laughing matter? (Re: [ccp4bb] process SeMet labelled data)
Greetings (or in correct Swiss German Grüezi wohl, with Umlaut=vowel mutation) to all CCP4BB subscribers, being a CCP4BB reader (and sometimes writer) since something like 15 years, I realized today that the comment of Gerard was the first one I read since then containing a side-swipe regarding the nationality or the national character of authors. I think, within a multinational scientific community with a very long lasting tradition of internationalism such comments should be beyond this forum. On the other hand Gerard's comment contains a somewhat humorous (Swiss, Swedish, German, Dutch, ... kind of?) aspect, since it refers partly to Switzerland, a country where many foreigners live and work, including Germans like Dirk. I would guess a situation similar like in Sweden or The Netherlands, isn't it? Sorry being German too I could not resist making such a moralizing comment. Coming back to the original question from Shivesh Kumar, I believe Dirk is right. Shivas' question was not very specific. He probably simply wanted to know how to process MAD data correctly e.g. regarding the anomalous signal, which resulted sometimes in sign problems when using CCP4 programs (at least with previous versions), if I remember it correctly. Such questions came up frequently in the past and were not answered in a nasty/sarcastic way. --cd Dr. Klaus Piontek Albert-Ludwigs-University Freiburg -ALU-FR- (ALU-FR is an equal opportunity employer dedicated to the goal of building a culturally diverse and pluralistic community with a multicultural environment) Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: [EMAIL PROTECTED] Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/ X-Real-To: [EMAIL PROTECTED] X-RAL-MFrom: [EMAIL PROTECTED] X-RAL-Connect: elvira.its.uu.se [130.238.164.5] X-CCLRC-SPAM-report: 0 : Date: Thu, 1 Mar 2007 10:31:06 +0100 Reply-To: Gerard DVD Kleywegt [EMAIL PROTECTED] Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK From: Gerard DVD Kleywegt [EMAIL PROTECTED] Subject: [ccp4bb] Swiss humour - no laughing matter? (Re: [ccp4bb] process SeMet labelled data) Comments: To: Dirk Kostrewa [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK List-Help: http://www.jiscmail.ac.uk/cgi-bin/webadmin?LIST=CCP4BB, mailto:[EMAIL PROTECTED] CCP4BB List-Unsubscribe: mailto:[EMAIL PROTECTED] List-Subscribe: mailto:[EMAIL PROTECTED] List-Owner: mailto:[EMAIL PROTECTED] List-Archive: http://www.jiscmail.ac.uk/cgi-bin/webadmin?LIST=CCP4BB gruzi dirk, come on - lighten up a little! your reply wasn't funny or helpful either - just moralising. (by the way - 't is a strange fact that whenever i get any negative reactions to my own postings they stem *exclusively* from any or all of these three countries: the us, germany and switzerland. positive reactions, on the other hand, are typically from (again) the us and the uk. i think there might be a phd thesis in there for some cultural anthropologist) to address your point - we are here to try and answer queries, not to teach people crystallography from the ground up. that's what supervisors are for. and my advice to students without (access to) crystallographically trained supervisors would be to apply for a good course (e.g., the one in cold spring harbor every autumn), to move elsewhere or to not do crystallography (so as to avoid mono-, di-, tri-, tetra- or pentaretractions, which tend to be a bit of a blot on anyone's cv) i think this is the point that the people who replied carefully etc. were making, albeit more succinctly and humourously (albeit a trifle sarcastically, perhaps). moreover, these replies came from people who have contributed seriously to ccp4bb on numerous occasions! look, we're all busy, and time-waster postings are annoying. such postings, and moralisations about good style, can eventually tee off the experts who take the time to share their knowledge and expertise. if joe bloggs, who never posts anything, is annoyed by one of my posts and unsubscribes, that's his loss. if, however, people like tassos unsubscribe it's *our* loss! so, dear subscribers: before posting a question - ask your supervisor and/or your colleagues. if they can't help you, search the web with google (in many cases your question will have been asked, and answered, before). if you're still not happy, then by all means post a message to the appropriate bulletin board darn! look what you've made me do! now i'm moralising myself! guess i'd better move to switzerland then ;-) --dvd On Thu, 1 Mar 2007, Dirk Kostrewa wrote: Hi Mark, although Shivesh's question was not very specific, and he should have clearly given some more informations about what he would like to know, he is probably a beginner in crystallography and simply asked
