Re: [ccp4bb] Protein crystallizes while concentration
I see some solutions to your problem and one works well for me on a small protein domain. I had exactly the same problem of crystallization during concentration and in my case I solve the problem by heating the crystal suspension. The validation of the protocol was made with support of 1D NMR to verify the stability of the structure after heating. So the solutions I see are: - Concentrate your sample until apparition of crystals and mildly heat it until solubilization of your crystals. Make a drop of 5µL in a crystallization plate and let it cool down against 500µL of your buffer. - Concentrate your sample until apparition of crystals and add the necessary amount of water to solubilize your crystals. Make drops of 1 or 2µL in a crystallization plate and let it concentrate against 500µL of your buffer. - You can also choose the best solution (heating or dilution) to solubilize your crystals ans try mild evaporation under paraffin oil to try to obtain crystals. And after, with the best solution, you can play with the salt concentration of reservoir solution to try to obtain better crystals. Gook luck. Michel. 2010/2/18 Daniel Ryan d.z.r...@dundee.ac.uk A cheaper solution than buying the dialysis buttons would be just to make your own out of the top of a microtube. Depending on what volume you want to dialyse you can use either a PCR tube (~35 uL per lid) or larger 1.5 mL tube (~250 uL). Just cut the top off the tube using a hot scalpel, you then place your sample in the cap of the microtube, cover it with dialysis membrane and then secure the tubing using the top part of the tube that you cut-off. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Lari Lehtiö Sent: 18 February 2010 16:38 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein crystallizes while concentration Dear Rajkumar, I would use dialysis buttons. E.g. http://hamptonresearch.com/product_detail.aspx?cid=10sid=63pid=111 Put your protein to the button and seal it with a piece of dialysis membrane. Place this to a linbro plate (easy to look with a microscope), fill the well with low salt buffer and seal with a cover slip. To make the diffusion slower, you can put the dialysis button inside a dialysis tubing with some high salt buffer and place this to a bigger volume of low salt buffer. ~L~ __ Lari Lehtiö Pharmacy, Department of Biochemistry and Pharmacy Åbo Akademi University, BioCity, FIN-20520 Turku Finland +358 2 215 4270 http://www.users.abo.fi/llehtio/ __ Quoting E rajakumar e_rajaku...@yahoo.com: Dear All I am Rajkumar, working on the protein which has unusual behavior while concentration. When I kept protein in 15 mM Tris 7.5, 150 mM NaCl and 5 mM DTT, the solubility of the protein is decreases drastically and tend to crystallize while concentration. Protein cannot be concentrated more than 3 mg/mL, however I noticed white turbid protein if I force to concentrate 3mg/mL. When I observed this white turbid solution under the microscope, I noticed shower of tiny protein crystals which are needle in shape. I screened freshly purified protein (2.5 mg/mL) in different Hampton and Qiagen screens, strangely none of the conditions gave the crystals. I concentrated left over protein at 15oC at 3 mg/mL and kept in the 4oC for 4 days again I noticed shower of crystals. This protein solubility is increased to ~20mg/mL when I kept in 15 Tris 7.5, 400 mM NaCl, 5% Glycerol and 5 mM DTT, but did not crystallize while concentration and also after screening with Hampton and Qiagen screens. My queries are 1. How do I get the crystals in the crystallization set up rather than while concentration, so that I can control the diffusion and finally nucleation? 2. Could anybody give me suggestions on seeding in this type of situation? 3. Any comments on reverse vapor diffusion for this type of protein are most welcome. So I can keep protein in high ionic strength (~400 mM NaCl)and diffuse against low Ionic strength or deionized water? Or any other protocol? Any suggestions are well appreciated. Thanking you in advance Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your new Email address! Grab the Email name you#39;ve always wanted before someone else does! http://mail.promotions.yahoo.com/newdomains/aa/
[ccp4bb] Position available at Diamond Light Source
Dear BBers I would like to draw your attention to a scientific instrumentation support post (http://www.diamond.ac.uk/Home/Jobs/Current/DIA0547.html) available at Diamond Light Source. Whilst this position is principally working for the tuneable MX beamline I04 (http://www.diamond.ac.uk/Home/Beamlines/MX/I04.html) the successful candidate will also contribute to the user programme and projects across the range of MX beamlines at Diamond (http://www.diamond.ac.uk/Home/Beamlines/MX.html). Best regards Dave Hall -- Principal Beamline Scientist Macromolecular Crystallography Diamond Light Source, Chilton, OX11 0DE, UK Tel: 00 44 12 35 77 89 26
Re: [ccp4bb] Expression of a protein of 43KDa
Hi In addition to what Fred and others have said, it is important to remember there are several native, stress-responsive proteins of E. coli that show good affinity for metal ions. Hence, they can be easily co-purified by immobilised metal affinity chromatography (IMAC). This seems particularly critical when the recombinant protein/protein domain of interest is expressed at very low levels and/or shows low metal binding capacity. A number of reasons for this include having the histidine tag partially buried, low intrinsic stability of your protein; its tendency to aggregate; etc.). We wrote a short review on this issue not long ago hoping it would help to identify quickly the usual suspects (doi:10.1016/j.bbagen.2006.03.027). I totally endorse what others have said: by all means always check the DNA sequence of what you want to express, and confirm its identity once you have purified it (N-terminal sequence, mass spec., etc.) to avoid disappointment and save resources. Kind regards Victor (Tom Blundell lab). Department of Biochemistry University of Cambridge 80 Tennis Court Road CB2 1GA Cambridge -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kerff Frédéric Sent: 18 February 2010 23:16 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Expression of a protein of 43KDa The maltose binding protein is one possibility. Some collaborators did provide us with very pure MBP instead of the protein of interest... Mass spec eventually identified it after we wasted some time on it... Fred I am trying to overexpress a His-tagged protein of 29KDa in E.coli (BL21-codon plus) and I end up with a highly expressed product that of 43KDa that binds to the Ni-column. I also have nice crystals. Does anyone have any experience on this. Armando
Re: [ccp4bb] phi/psi angle display in coot
Ethan Merritt wrote: On Thursday 18 February 2010 14:48:57 Paul Emsley wrote: Ursula Schulze-Gahmen wrote: I am a new user of Coot and I haven't found an easy way of displaying the phi/psi angle of a specific residue while I am rebuilding. I found of course the Ramachandran plot, but i would like a way to display the main chain geometry of a specific residue. You can't do it :-/. (not that it's any help, it's been requested before - it is on the list of things to do.) Huh? In my experience when you select the residue in the main window, the corresponding mark in the Ramachandran diagram lights up in green. If you rebuild it and select again, the green-lit mark moves accordingly. Do I have a magically-enhanced version of the program? :-) Perhaps I misunderstood the question then. I took it to mean: what are the phi and psi value of the residue that I click on?. Those values are, as you say, represented on a Ramachandran plot with a green block. In due course the values will be added to the residue info dialog. Paul.
[ccp4bb] PEG interaction with Trp in active site
Hello, sorry for my off topic question. I found a PEG molecule bound to the active site of my enzyme structure. And I did not expected it there, though I used PEG as precipitant in the crystallization condition. Now I'm wondering how could bind PEG with its hydrophobic nature at all in the hydrophobic active site? It seems that the PEG molecule interacts predominantly with Tryptophans (distance 3.8 A). Did anybody observe similar cases? And which kind of interaction should there be active? I would be grateful for your help, cheers, Marek __ Do You Yahoo!? Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden Schutz gegen Massenmails. http://mail.yahoo.com
Re: [ccp4bb] anomalous difference fourier maps
Dear all, This is a remark I have wanted to make for a long time but managed so far to repress. However, this case is absolutely clear: Ivan was not asking for general advice on how to carry out a general task, but how to perform a specific task with the CCP4 software. In response we get (surprise, surprise, ...) another instance of the relentless touting for Phenix on the CCP4BB, which has long been an expected (or tolerated?) feature of this BB. Contributions from Phenix developers are of course much appreciated when questions are about general crystallographic matters where their expertise and experience is valuable; but when people ask specifically how to do something with CCP4 programs, could they please not be grabbed by the sleeve and enticed to buy their sweets from the shop next door? In this case, for instance, Ivan thanks guys (plural) for the answers he got (All of your suggestions were great). Perhaps one of those was a CCP4-based answer, but if so it has not even been communicated to the rest of the CCP4BB subscribers - so not only is this touting in bad taste after a while: it even interferes with the sharing of expertise in using the CCP4 software, which after all must be one of the main missions of this BB. I have long wondered whether anyone on the CCP4 side been assigned the task of answering every question to the Phenix BB by describing how to do it with CCP4 programs ... . With best wishes, Gerard. -- On Thu, Feb 18, 2010 at 03:53:02PM -0800, Pavel Afonine wrote: Hi Ivan, two ways (at least) to do it in PHENIX: - phenix.refine always computes anomalous difference Fourier map (provided that your input data file contains Fobs(+) and Fobs(-)). The command below will do it: phenix.refine model.pdb data.mtz strategy=none main.number_of_macro_cycles=0 output.prefix=maps_only - you can use phenix.maps that is a general tool to compute a broad variety of maps. Type phenix.maps from the command line for usage instructions. You need to have the latest development (or one of) PHENIX nightly build for this. All this is available from the GUI too. Pavel. On 2/18/10 3:34 PM, xaravich ivan wrote: Hello, I wanted to make an anomalous difference fourier map of a structure with zinc bound to it. However I have not been successful in making the map and I would really appreciate your help if anyone could suggest me where I am going wrong. I solved this zinc bound structure, by molecular replacement from a calcium bound structure (1.4 angstrom) that I solved. I want to show that the zinc binds to the identical site by the anomalous difference fourier map. I am using CCP4i and the steps that I have been taking are, (names of the files are arbitrary) 1) generating structure factors and phases from the solved coordinates by SFALL Input files zinc bound pdb original zinc .mtz data from synchrotron output file sfall.mtz 2)merging the sfall.mtz containing the PHICalc and FCalc columns with the original synchrotron .mtz file containing DANO and SIGDANO by running CAD. input files sfall.mtz and zinc synchrtron .mtz output file CAD.mtz 3) Running FFT to make anomalous map, selecting labels from CAD.mtz as input files. There is an output map file but nothing in it. all the values are 0 and the map is not recognized by coot. There is no error message in the log file. I must be missing something or doing something wrong/stupid. Thanks, Ivan -- === * * * 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 * * * ===
[ccp4bb] The X6A workbench winter session
The X6A workbench: Advanced Structural Biology Tools (http://protein.nsls.bnl.gov) We would like to invite you to participate in this four day hands-on course at the NIGMS Research Facility at the National Synchrotron Light Source. This course is intended for beginners who would like to obtain an overview on macromolecular structure determination. This course will discuss the basic concepts of macromolecular crystallography beam lines at synchrotron facilities, crystal growth and handling, data collection and processing, phasing and the first steps in refinement. Most time will be spend at the beam line actually handling crystals and screening for cryoprotectants, collecting and processing data. Significant amount of time is spent phasing and obtaining the first electron density map. Registration; http://protein.nsls.bnl.gov; you must have an active guest appointment at Brookhaven National Laboratory at the time of the course.
Re: [ccp4bb] anomalous difference fourier maps
Dear Gerard, I can only agree with you - I've also noticed a growing and sometimes irritating cross-advertisement of non-CCP4 programs on the CCP4BB over the last months (mainly Phenix). Unless, the specific task that was asked for, can only be (reasonably) solved with non-CCP4 programs, such replies leave a somewhat bad aftertaste. Personally, I think, it would be perfectly acceptable if both solutions with CCP4 programs and other programs would be given, so that the user may choose, or try them all. Best wishes, Dirk. Am 19.02.10 15:04, schrieb Gerard Bricogne: Dear all, This is a remark I have wanted to make for a long time but managed so far to repress. However, this case is absolutely clear: Ivan was not asking for general advice on how to carry out a general task, but how to perform a specific task with the CCP4 software. In response we get (surprise, surprise, ...) another instance of the relentless touting for Phenix on the CCP4BB, which has long been an expected (or tolerated?) feature of this BB. Contributions from Phenix developers are of course much appreciated when questions are about general crystallographic matters where their expertise and experience is valuable; but when people ask specifically how to do something with CCP4 programs, could they please not be grabbed by the sleeve and enticed to buy their sweets from the shop next door? In this case, for instance, Ivan thanks guys (plural) for the answers he got (All of your suggestions were great). Perhaps one of those was a CCP4-based answer, but if so it has not even been communicated to the rest of the CCP4BB subscribers - so not only is this touting in bad taste after a while: it even interferes with the sharing of expertise in using the CCP4 software, which after all must be one of the main missions of this BB. I have long wondered whether anyone on the CCP4 side been assigned the task of answering every question to the Phenix BB by describing how to do it with CCP4 programs ... . With best wishes, Gerard. -- On Thu, Feb 18, 2010 at 03:53:02PM -0800, Pavel Afonine wrote: Hi Ivan, two ways (at least) to do it in PHENIX: - phenix.refine always computes anomalous difference Fourier map (provided that your input data file contains Fobs(+) and Fobs(-)). The command below will do it: phenix.refine model.pdb data.mtz strategy=none main.number_of_macro_cycles=0 output.prefix=maps_only - you can use phenix.maps that is a general tool to compute a broad variety of maps. Type phenix.maps from the command line for usage instructions. You need to have the latest development (or one of) PHENIX nightly build for this. All this is available from the GUI too. Pavel. On 2/18/10 3:34 PM, xaravich ivan wrote: Hello, I wanted to make an anomalous difference fourier map of a structure with zinc bound to it. However I have not been successful in making the map and I would really appreciate your help if anyone could suggest me where I am going wrong. I solved this zinc bound structure, by molecular replacement from a calcium bound structure (1.4 angstrom) that I solved. I want to show that the zinc binds to the identical site by the anomalous difference fourier map. I am using CCP4i and the steps that I have been taking are, (names of the files are arbitrary) 1) generating structure factors and phases from the solved coordinates by SFALL Input files zinc bound pdb original zinc .mtz data from synchrotron output file sfall.mtz 2)merging the sfall.mtz containing the PHICalc and FCalc columns with the original synchrotron .mtz file containing DANO and SIGDANO by running CAD. input files sfall.mtz and zinc synchrtron .mtz output file CAD.mtz 3) Running FFT to make anomalous map, selecting labels from CAD.mtz as input files. There is an output map file but nothing in it. all the values are 0 and the map is not recognized by coot. There is no error message in the log file. I must be missing something or doing something wrong/stupid. Thanks, Ivan -- *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] anomalous difference fourier maps
Hi Dirk, When it happens that I reply to a ccp4bb message and that the answer, or solution I may have (which I think is better or more appropriate) involves using non-ccp4 programs, I do it off-list. By replying privately to the person who asked the question. Fred. Dirk Kostrewa wrote: Dear Gerard, I can only agree with you - I've also noticed a growing and sometimes irritating cross-advertisement of non-CCP4 programs on the CCP4BB over the last months (mainly Phenix). Unless, the specific task that was asked for, can only be (reasonably) solved with non-CCP4 programs, such replies leave a somewhat bad aftertaste. Personally, I think, it would be perfectly acceptable if both solutions with CCP4 programs and other programs would be given, so that the user may choose, or try them all. Best wishes, Dirk.
Re: [ccp4bb] anomalous difference fourier maps
I am inclined to agree with Gerard. Of course if there is a specific question to CCP4bb about SHELX, I try to answer it. Since I am too lazy to maintain my own bb, this is very convenient. However I have stopped 'poaching', for example for the thread in question I resisted the temptation to point out that SHELXE has a convenient way of making such anomalous maps, because that would not have been a direct answer to the question and CCP4 provides similar facilities (and SHARP would be even better). We use the CCP4 programs (especially REFMAC and COOT) extensively and are very grateful for all the support we get. In particular, we should not underestimate the unique role CCP4bb plays in crystallographic education. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Fri, 19 Feb 2010, Gerard Bricogne wrote: Dear all, This is a remark I have wanted to make for a long time but managed so far to repress. However, this case is absolutely clear: Ivan was not asking for general advice on how to carry out a general task, but how to perform a specific task with the CCP4 software. In response we get (surprise, surprise, ...) another instance of the relentless touting for Phenix on the CCP4BB, which has long been an expected (or tolerated?) feature of this BB. Contributions from Phenix developers are of course much appreciated when questions are about general crystallographic matters where their expertise and experience is valuable; but when people ask specifically how to do something with CCP4 programs, could they please not be grabbed by the sleeve and enticed to buy their sweets from the shop next door? In this case, for instance, Ivan thanks guys (plural) for the answers he got (All of your suggestions were great). Perhaps one of those was a CCP4-based answer, but if so it has not even been communicated to the rest of the CCP4BB subscribers - so not only is this touting in bad taste after a while: it even interferes with the sharing of expertise in using the CCP4 software, which after all must be one of the main missions of this BB. I have long wondered whether anyone on the CCP4 side been assigned the task of answering every question to the Phenix BB by describing how to do it with CCP4 programs ... . With best wishes, Gerard. -- On Thu, Feb 18, 2010 at 03:53:02PM -0800, Pavel Afonine wrote: Hi Ivan, two ways (at least) to do it in PHENIX: - phenix.refine always computes anomalous difference Fourier map (provided that your input data file contains Fobs(+) and Fobs(-)). The command below will do it: phenix.refine model.pdb data.mtz strategy=none main.number_of_macro_cycles=0 output.prefix=maps_only - you can use phenix.maps that is a general tool to compute a broad variety of maps. Type phenix.maps from the command line for usage instructions. You need to have the latest development (or one of) PHENIX nightly build for this. All this is available from the GUI too. Pavel. On 2/18/10 3:34 PM, xaravich ivan wrote: Hello, I wanted to make an anomalous difference fourier map of a structure with zinc bound to it. However I have not been successful in making the map and I would really appreciate your help if anyone could suggest me where I am going wrong. I solved this zinc bound structure, by molecular replacement from a calcium bound structure (1.4 angstrom) that I solved. I want to show that the zinc binds to the identical site by the anomalous difference fourier map. I am using CCP4i and the steps that I have been taking are, (names of the files are arbitrary) 1) generating structure factors and phases from the solved coordinates by SFALL Input files zinc bound pdb original zinc .mtz data from synchrotron output file sfall.mtz 2)merging the sfall.mtz containing the PHICalc and FCalc columns with the original synchrotron .mtz file containing DANO and SIGDANO by running CAD. input files sfall.mtz and zinc synchrtron .mtz output file CAD.mtz 3) Running FFT to make anomalous map, selecting labels from CAD.mtz as input files. There is an output map file but nothing in it. all the values are 0 and the map is not recognized by coot. There is no error message in the log file. I must be missing something or doing something wrong/stupid. Thanks, Ivan -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * *
Re: [ccp4bb] Expression of a protein of 43KDa
Dear Armando, Two similar, but not identical experiences: - we expressed a protein and got some ugly small crystals. Then the Edman degradation results on the purified protein came in and turned out to correspond to chloramphenicol transferase (same size as our protein). - expressed a protein and got very nice crystals, got a dataset with cell parameters suspiciously like LyCl7 (lysozyme heptachloride, that protein that crystallises as easily as NaCl). Solved the structure and of course it WAS LyCl7. The LyCl7 was a minor fraction in the protein prep., but still crystallised (also same size as our protein). Fortunately, in both cases we only lost several weeks and this is only a small percentage of our failures. Also, they are remediable ones, most failed projects are because the protein does not express in soluble form, aggregates aspecifically or simply refuses to crystallise... In summary, like others, I can only stress the importance of checking by N-terminal sequence analysis, mass-spec, Western-blotting etc. that that band on the gel is indeed the protein you want. Greetings, Mark PS CIB in Madrid has a proteomics service we use. Becaus it belongs to CSIC, they will charge you the internal tariff: http://www.cib.csic.es/en/servicio.php?iddepartamento=27 Mark J. van Raaij http://webspersoais.usc.es/mark.vanraaij/ researcherID: B-3678-2009 On 18 Feb 2010, at 18:49, Armando Albert de la Cruz wrote: I am trying to overexpress a His-tagged protein of 29KDa in E.coli (BL21-codon plus) and I end up with a highly expressed product that of 43KDa that binds to the Ni-column. I also have nice crystals. Does anyone have any experience on this. Armando
[ccp4bb] domain contact surface
Dear all, I am trying to calculate the domain-domain contact surface from one chain. I see AREAIMOL to only tell you the accessible surface area/residue. Could any other software/webserve could specify residues in the contact surface and calculate the total contact area? Thanks J.
Re: [ccp4bb] anomalous difference fourier maps
The guidelines for the CCP4BB are extremely broad and certainly include discussion of other software packages. Since the original poster's question had to do with a specific problem with CCP4, it would have been appropriate for Pavel to prefix his reply with something like I hope you receive an answer to your question shortly, but in the meantime here is an alternative. But this is a nicety I would be glad to forgo for the sake of getting the extra information. The problem is not that the phenix team was so quick to promote their software, but rather that now 14 hours after the original post, no one has answered the CCP4 question. It is too easy to say yes, I think I could help this guy, but half the readers of this BB could probably give a better answer. Maybe someone at CCP4 should be assigned to answer all reasonable queries that go unanswered for more than 8 hours. The phenomenal rise in popularity of the phenix package is probably due as much to the incredible responsiveness of the phenix team, not only in support but in adding requested features, as it is to the power and ease of use of the programs. Vellieux Frederic wrote: Hi Dirk, When it happens that I reply to a ccp4bb message and that the answer, or solution I may have (which I think is better or more appropriate) involves using non-ccp4 programs, I do it off-list. By replying privately to the person who asked the question. Fred. Dirk Kostrewa wrote: Dear Gerard, I can only agree with you - I've also noticed a growing and sometimes irritating cross-advertisement of non-CCP4 programs on the CCP4BB over the last months (mainly Phenix). Unless, the specific task that was asked for, can only be (reasonably) solved with non-CCP4 programs, such replies leave a somewhat bad aftertaste. Personally, I think, it would be perfectly acceptable if both solutions with CCP4 programs and other programs would be given, so that the user may choose, or try them all. Best wishes, Dirk.
Re: [ccp4bb] domain contact surface
You would have to cheat a little bit, and relabel the second domain chain id to something different. Or you can split the pdb file into two. That would still be necessary for using the EBI PISA server at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html as I have just been doing. In my case I wanted the interface between multi- chain domains, so I had to pretend they were one chain each. It works alright. Pierre Dr. Pierre Rizkallah, Senior Lecturer in Structural Biology, WHRI, School of Medicine, Cardiff University, Heath Park, CF14 4XN email: rizkall...@cf.ac.uk phone + 44 29 2074 2248 Jane Bailey jbailey.x...@googlemail.com 19/02/10 3:44 PM Dear all, I am trying to calculate the domain-domain contact surface from one chain. I see AREAIMOL to only tell you the accessible surface area/residue. Could any other software/webserve could specify residues in the contact surface and calculate the total contact area? Thanks J.
Re: [ccp4bb] PEG interaction with Trp in active site
Is this the active enzyme or an inactive mutant? does your substrate have any similarity with PEG (size, conformation, bulk etc?) would you assume that if there was a substrate bound to the active site and say a few waters, and/or metal ions it would probably fill the space which in this case PEG is taking. I think it would be more work, but the best way would be to try to crystallize a inactive mutant with very less change in the active site residues with the substrate( or active enzyme with an analog) and see if the PEG is displaced. ivan On Fri, Feb 19, 2010 at 3:42 AM, Marek Frischerkase frischerkasema...@yahoo.de wrote: Hello, sorry for my off topic question. I found a PEG molecule bound to the active site of my enzyme structure. And I did not expected it there, though I used PEG as precipitant in the crystallization condition. Now I'm wondering how could bind PEG with its hydrophobic nature at all in the hydrophobic active site? It seems that the PEG molecule interacts predominantly with Tryptophans (distance 3.8 A). Did anybody observe similar cases? And which kind of interaction should there be active? I would be grateful for your help, cheers, Marek __ Do You Yahoo!? Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden Schutz gegen Massenmails. http://mail.yahoo.com
Re: [ccp4bb] [ccp4] domain contact surface
Dear Jane, try to split your protein into two chains (e.g. domain 1 -- chain A / domain 2 -- chain B in a PDB file) and use the PISA server for processing. Besides several interfaces with symmetry mates, one should represent your domain/domain interface based on chain A/B you've chosen. Greetings, Matthias _ http://redirect.gimas.net/?n=M1002xHMNoSpam2 Keine Lust auf Spam? Hotmail blockiert Spam automatisch
Re: [ccp4bb] anomalous difference fourier maps
On Fri, 2010-02-19 at 10:37 -0500, Edward A. Berry wrote: The problem is not that the phenix team was so quick to promote their software, but rather that now 14 hours after the original post, no one has answered the CCP4 question. The original poster returned to indicate that he had received multiple responses. I gather that he received them off-board. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] was: anomalous difference fourier maps
Hi Everyone, my apologies for those who got irritated. I guess when someone got stuck with something and asks for help, he/she deserves a solution for the problem. Providing with a solution is what I have in mind replying to the questions. The only reason why I reply to the whole list and not personally is because typically the similar questions may come in the future again and replying to the list will save my time by contributing to the archive instead of replying to the same question personally multiple times. The line between addressing the questions and thinking who can get upset about it (apart from the person who asked for help), is subtle and I typically don't have time to think about it, sorry. Being a non-native English speaker, decorating my emails by prefixing them with nice words is hard for me and time inefficient - so I apologies for this now and in advance. The CCP4bb is the great forum for addressing/discussing various crystallographic questions and tools, and I believe the everyone will benefit if it remains the same in future. Have a good week-end, Pavel. On 2/19/10 6:57 AM, George M. Sheldrick wrote: I am inclined to agree with Gerard. Of course if there is a specific question to CCP4bb about SHELX, I try to answer it. Since I am too lazy to maintain my own bb, this is very convenient. However I have stopped 'poaching', for example for the thread in question I resisted the temptation to point out that SHELXE has a convenient way of making such anomalous maps, because that would not have been a direct answer to the question and CCP4 provides similar facilities (and SHARP would be even better). We use the CCP4 programs (especially REFMAC and COOT) extensively and are very grateful for all the support we get. In particular, we should not underestimate the unique role CCP4bb plays in crystallographic education. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Fri, 19 Feb 2010, Gerard Bricogne wrote: Dear all, This is a remark I have wanted to make for a long time but managed so far to repress. However, this case is absolutely clear: Ivan was not asking for general advice on how to carry out a general task, but how to perform a specific task with the CCP4 software. In response we get (surprise, surprise, ...) another instance of the relentless touting for Phenix on the CCP4BB, which has long been an expected (or tolerated?) feature of this BB. Contributions from Phenix developers are of course much appreciated when questions are about general crystallographic matters where their expertise and experience is valuable; but when people ask specifically how to do something with CCP4 programs, could they please not be grabbed by the sleeve and enticed to buy their sweets from the shop next door? In this case, for instance, Ivan thanks guys (plural) for the answers he got (All of your suggestions were great). Perhaps one of those was a CCP4-based answer, but if so it has not even been communicated to the rest of the CCP4BB subscribers - so not only is this touting in bad taste after a while: it even interferes with the sharing of expertise in using the CCP4 software, which after all must be one of the main missions of this BB. I have long wondered whether anyone on the CCP4 side been assigned the task of answering every question to the Phenix BB by describing how to do it with CCP4 programs ... . With best wishes, Gerard. -- On Thu, Feb 18, 2010 at 03:53:02PM -0800, Pavel Afonine wrote: Hi Ivan, two ways (at least) to do it in PHENIX: - phenix.refine always computes anomalous difference Fourier map (provided that your input data file contains Fobs(+) and Fobs(-)). The command below will do it: phenix.refine model.pdb data.mtz strategy=none main.number_of_macro_cycles=0 output.prefix=maps_only - you can use phenix.maps that is a general tool to compute a broad variety of maps. Type phenix.maps from the command line for usage instructions. You need to have the latest development (or one of) PHENIX nightly build for this. All this is available from the GUI too. Pavel. On 2/18/10 3:34 PM, xaravich ivan wrote: Hello, I wanted to make an anomalous difference fourier map of a structure with zinc bound to it. However I have not been successful in making the map and I would really appreciate your help if anyone could suggest me where I am going wrong. I solved this zinc bound structure, by molecular replacement from a calcium bound structure (1.4 angstrom) that I solved. I want to show that the zinc binds to the identical site by the anomalous difference fourier map. I am using CCP4i and the steps that I have been taking are, (names of the files are arbitrary) 1) generating structure factors and phases from the solved coordinates
Re: [ccp4bb] anomalous difference fourier maps, or how to keep chastity of CCP4BB
I guess Phenix people are just trying to help. After all they are not selling us Zinger sewing machines (zin...@sewing CO, my apology) nor they are trying to push us Kirby (http://www.consumeraffairs.com/in_home/kirby.htm, no apology) vacuum cleaners using naive chastity of CCP4BB site. They are a bunch of young dynamic people, all of them excellent crystallographers, who are participate in evolution of the alternative tool for crystallographic calculations with already possess visible nice features. They trespass borders of CCP4BB quite frequently, but for that they don't have to be prosecuted. However CCP4BB subscribers can start discussion about implementation of chastity belt (see Wiki site http://en.wikipedia.org/wiki/Chastity_belt for explanation of chastity belt metaphor) for prevention of CCP4BB abuse. Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Feb 19, 2010, at 16:04 , Gerard Bricogne wrote: Dear all, This is a remark I have wanted to make for a long time but managed so far to repress. However, this case is absolutely clear: Ivan was not asking for general advice on how to carry out a general task, but how to perform a specific task with the CCP4 software. In response we get (surprise, surprise, ...) another instance of the relentless touting for Phenix on the CCP4BB, which has long been an expected (or tolerated?) feature of this BB. Contributions from Phenix developers are of course much appreciated when questions are about general crystallographic matters where their expertise and experience is valuable; but when people ask specifically how to do something with CCP4 programs, could they please not be grabbed by the sleeve and enticed to buy their sweets from the shop next door? In this case, for instance, Ivan thanks guys (plural) for the answers he got (All of your suggestions were great). Perhaps one of those was a CCP4-based answer, but if so it has not even been communicated to the rest of the CCP4BB subscribers - so not only is this touting in bad taste after a while: it even interferes with the sharing of expertise in using the CCP4 software, which after all must be one of the main missions of this BB. I have long wondered whether anyone on the CCP4 side been assigned the task of answering every question to the Phenix BB by describing how to do it with CCP4 programs ... . With best wishes, Gerard. -- On Thu, Feb 18, 2010 at 03:53:02PM -0800, Pavel Afonine wrote: Hi Ivan, two ways (at least) to do it in PHENIX: - phenix.refine always computes anomalous difference Fourier map (provided that your input data file contains Fobs(+) and Fobs(-)). The command below will do it: phenix.refine model.pdb data.mtz strategy=none main.number_of_macro_cycles=0 output.prefix=maps_only - you can use phenix.maps that is a general tool to compute a broad variety of maps. Type phenix.maps from the command line for usage instructions. You need to have the latest development (or one of) PHENIX nightly build for this. All this is available from the GUI too. Pavel. On 2/18/10 3:34 PM, xaravich ivan wrote: Hello, I wanted to make an anomalous difference fourier map of a structure with zinc bound to it. However I have not been successful in making the map and I would really appreciate your help if anyone could suggest me where I am going wrong. I solved this zinc bound structure, by molecular replacement from a calcium bound structure (1.4 angstrom) that I solved. I want to show that the zinc binds to the identical site by the anomalous difference fourier map. I am using CCP4i and the steps that I have been taking are, (names of the files are arbitrary) 1) generating structure factors and phases from the solved coordinates by SFALL Input files zinc bound pdb original zinc .mtz data from synchrotron output file sfall.mtz 2)merging the sfall.mtz containing the PHICalc and FCalc columns with the original synchrotron .mtz file containing DANO and SIGDANO by running CAD. input files sfall.mtz and zinc synchrtron .mtz output file CAD.mtz 3) Running FFT to make anomalous map, selecting labels from CAD.mtz as input files. There is an output map file but nothing in it. all the values are 0 and the map is not recognized by coot. There is no error message in the log file. I must be missing something or doing something wrong/stupid. Thanks, Ivan -- === * * * Gerard Bricogne
Re: [ccp4bb] PEG interaction with Trp in active site
Any chance that it is myristate or some other fatty acid? Good luck- Steve On Fri, Feb 19, 2010 at 3:42 AM, Marek Frischerkase frischerkasema...@yahoo.de wrote: Hello, sorry for my off topic question. I found a PEG molecule bound to the active site of my enzyme structure. And I did not expected it there, though I used PEG as precipitant in the crystallization condition. Now I'm wondering how could bind PEG with its hydrophobic nature at all in the hydrophobic active site? It seems that the PEG molecule interacts predominantly with Tryptophans (distance 3.8 A). Did anybody observe similar cases? And which kind of interaction should there be active? I would be grateful for your help, cheers, Marek __ Do You Yahoo!? Sie sind Spam leid? Yahoo! Mail verfügt über einen herausragenden Schutz gegen Massenmails. http://mail.yahoo.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] anomalous difference fourier maps
ok I think I should say something here.For some reason I was unable to find REPLY-ALL button and my reply did not go to everyone first so I had to write another message.I got the answer to my original query and I used CCP4 (CAD) and coot as suggested by Jan. Having said that I did not know that you could do it in so many ways. and that is perhaps the most valuable thing!!! Thanks all of you for your replies and I think the best thing of this forum is to be able to get answers when you do not have anyone in the lab to guide you. People like us need you guys the most. Irrespective of whether it is ccp4bb or phenix or any other program, the goal is to share the knowledge and help the less experienced (less fortunate) with your vast expertise, so that people can enjoy doing exciting crystallography related research. Thanks to everyone, Ivan On Fri, Feb 19, 2010 at 7:37 AM, Edward A. Berry ber...@upstate.edu wrote: The guidelines for the CCP4BB are extremely broad and certainly include discussion of other software packages. Since the original poster's question had to do with a specific problem with CCP4, it would have been appropriate for Pavel to prefix his reply with something like I hope you receive an answer to your question shortly, but in the meantime here is an alternative. But this is a nicety I would be glad to forgo for the sake of getting the extra information. The problem is not that the phenix team was so quick to promote their software, but rather that now 14 hours after the original post, no one has answered the CCP4 question. It is too easy to say yes, I think I could help this guy, but half the readers of this BB could probably give a better answer. Maybe someone at CCP4 should be assigned to answer all reasonable queries that go unanswered for more than 8 hours. The phenomenal rise in popularity of the phenix package is probably due as much to the incredible responsiveness of the phenix team, not only in support but in adding requested features, as it is to the power and ease of use of the programs. Vellieux Frederic wrote: Hi Dirk, When it happens that I reply to a ccp4bb message and that the answer, or solution I may have (which I think is better or more appropriate) involves using non-ccp4 programs, I do it off-list. By replying privately to the person who asked the question. Fred. Dirk Kostrewa wrote: Dear Gerard, I can only agree with you - I've also noticed a growing and sometimes irritating cross-advertisement of non-CCP4 programs on the CCP4BB over the last months (mainly Phenix). Unless, the specific task that was asked for, can only be (reasonably) solved with non-CCP4 programs, such replies leave a somewhat bad aftertaste. Personally, I think, it would be perfectly acceptable if both solutions with CCP4 programs and other programs would be given, so that the user may choose, or try them all. Best wishes, Dirk.
Re: [ccp4bb] anomalous difference fourier maps
I guess it is a BB hosted by CCP4, but I think it serves a much wider community of structural biologists, and it is valuable for that community that there are suggestions about how to solve problems using SHELX, or BUSTER or PHENIX software.. Certainly I learn new tricks. So speaking as a CCP4 contributer lets keep it as broad based as possible. Maybe we should subscribe to the PHENIX BB and suggest alternative solutions there! Eleanor Dodson George M. Sheldrick wrote: I am inclined to agree with Gerard. Of course if there is a specific question to CCP4bb about SHELX, I try to answer it. Since I am too lazy to maintain my own bb, this is very convenient. However I have stopped 'poaching', for example for the thread in question I resisted the temptation to point out that SHELXE has a convenient way of making such anomalous maps, because that would not have been a direct answer to the question and CCP4 provides similar facilities (and SHARP would be even better). We use the CCP4 programs (especially REFMAC and COOT) extensively and are very grateful for all the support we get. In particular, we should not underestimate the unique role CCP4bb plays in crystallographic education. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Fri, 19 Feb 2010, Gerard Bricogne wrote: Dear all, This is a remark I have wanted to make for a long time but managed so far to repress. However, this case is absolutely clear: Ivan was not asking for general advice on how to carry out a general task, but how to perform a specific task with the CCP4 software. In response we get (surprise, surprise, ...) another instance of the relentless touting for Phenix on the CCP4BB, which has long been an expected (or tolerated?) feature of this BB. Contributions from Phenix developers are of course much appreciated when questions are about general crystallographic matters where their expertise and experience is valuable; but when people ask specifically how to do something with CCP4 programs, could they please not be grabbed by the sleeve and enticed to buy their sweets from the shop next door? In this case, for instance, Ivan thanks guys (plural) for the answers he got (All of your suggestions were great). Perhaps one of those was a CCP4-based answer, but if so it has not even been communicated to the rest of the CCP4BB subscribers - so not only is this touting in bad taste after a while: it even interferes with the sharing of expertise in using the CCP4 software, which after all must be one of the main missions of this BB. I have long wondered whether anyone on the CCP4 side been assigned the task of answering every question to the Phenix BB by describing how to do it with CCP4 programs ... . With best wishes, Gerard. -- On Thu, Feb 18, 2010 at 03:53:02PM -0800, Pavel Afonine wrote: Hi Ivan, two ways (at least) to do it in PHENIX: - phenix.refine always computes anomalous difference Fourier map (provided that your input data file contains Fobs(+) and Fobs(-)). The command below will do it: phenix.refine model.pdb data.mtz strategy=none main.number_of_macro_cycles=0 output.prefix=maps_only - you can use phenix.maps that is a general tool to compute a broad variety of maps. Type phenix.maps from the command line for usage instructions. You need to have the latest development (or one of) PHENIX nightly build for this. All this is available from the GUI too. Pavel. On 2/18/10 3:34 PM, xaravich ivan wrote: Hello, I wanted to make an anomalous difference fourier map of a structure with zinc bound to it. However I have not been successful in making the map and I would really appreciate your help if anyone could suggest me where I am going wrong. I solved this zinc bound structure, by molecular replacement from a calcium bound structure (1.4 angstrom) that I solved. I want to show that the zinc binds to the identical site by the anomalous difference fourier map. I am using CCP4i and the steps that I have been taking are, (names of the files are arbitrary) 1) generating structure factors and phases from the solved coordinates by SFALL Input files zinc bound pdb original zinc .mtz data from synchrotron output file sfall.mtz 2)merging the sfall.mtz containing the PHICalc and FCalc columns with the original synchrotron .mtz file containing DANO and SIGDANO by running CAD. input files sfall.mtz and zinc synchrtron .mtz output file CAD.mtz 3) Running FFT to make anomalous map, selecting labels from CAD.mtz as input files. There is an output map file but nothing in it. all the values are 0 and the map is not recognized by coot. There is no error message in the log file. I must be missing something or doing something wrong/stupid. Thanks, Ivan --
Re: [ccp4bb] domain contact surface
Ed Pozharski wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html Try PISA server - it will identify contacts and report total buried surface area per contact. On Fri, 2010-02-19 at 16:42 +0100, Jane Bailey wrote: Dear all, I am trying to calculate the domain-domain contact surface from one chain. I see AREAIMOL to only tell you the accessible surface area/residue. Could any other software/webserve could specify residues in the contact surface and calculate the total contact area? Thanks J. Dear all, Thanks very much for the answers. I managed to get the interface from PISA by assign two chain IDs onto two domains, as suggested by Pierre and Amy. It gave the interface Solvent-accessible area of each domain: 274.2, 312.2, but not the BSA. However I saw it gave the BSA of individual residue from the interface. Do people get the total interface BSA by adding them up? best Jane
Re: [ccp4bb] Protein crystallizes while concentration
Dear Daniel, You can also try adding 100-200 mM ammonium hydroxide. The jump in pH will dissolve your crystals. If you then set up your protein drop above the buffer of your liking, vapor diffusion will bring the pH back down to the original value, and in the process hopefully lead to more controlled nucleation and crystal growth. Worth to try on a few microliter of your prep. Han Han Remaut, PhD Structural Molecular Microbiology - VIB/VUB Building E, 4th Floor Pleinlaan 2 1050 Brussel email: han.rem...@vib-vub.be tel: +32-2-629 1923 / +32-499 708050 http://www.structuralbiology.be/research/adhesins On 19 Feb 2010, at 09:33, M T wrote: I see some solutions to your problem and one works well for me on a small protein domain. I had exactly the same problem of crystallization during concentration and in my case I solve the problem by heating the crystal suspension. The validation of the protocol was made with support of 1D NMR to verify the stability of the structure after heating. So the solutions I see are: - Concentrate your sample until apparition of crystals and mildly heat it until solubilization of your crystals. Make a drop of 5µL in a crystallization plate and let it cool down against 500µL of your buffer. - Concentrate your sample until apparition of crystals and add the necessary amount of water to solubilize your crystals. Make drops of 1 or 2µL in a crystallization plate and let it concentrate against 500µL of your buffer. - You can also choose the best solution (heating or dilution) to solubilize your crystals ans try mild evaporation under paraffin oil to try to obtain crystals. And after, with the best solution, you can play with the salt concentration of reservoir solution to try to obtain better crystals. Gook luck. Michel. 2010/2/18 Daniel Ryan d.z.r...@dundee.ac.uk A cheaper solution than buying the dialysis buttons would be just to make your own out of the top of a microtube. Depending on what volume you want to dialyse you can use either a PCR tube (~35 uL per lid) or larger 1.5 mL tube (~250 uL). Just cut the top off the tube using a hot scalpel, you then place your sample in the cap of the microtube, cover it with dialysis membrane and then secure the tubing using the top part of the tube that you cut-off. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Lari Lehtiö Sent: 18 February 2010 16:38 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein crystallizes while concentration Dear Rajkumar, I would use dialysis buttons. E.g. http://hamptonresearch.com/product_detail.aspx?cid=10sid=63pid=111 Put your protein to the button and seal it with a piece of dialysis membrane. Place this to a linbro plate (easy to look with a microscope), fill the well with low salt buffer and seal with a cover slip. To make the diffusion slower, you can put the dialysis button inside a dialysis tubing with some high salt buffer and place this to a bigger volume of low salt buffer. ~L~ __ Lari Lehtiö Pharmacy, Department of Biochemistry and Pharmacy Åbo Akademi University, BioCity, FIN-20520 Turku Finland +358 2 215 4270 http://www.users.abo.fi/llehtio/ __ Quoting E rajakumar e_rajaku...@yahoo.com: Dear All I am Rajkumar, working on the protein which has unusual behavior while concentration. When I kept protein in 15 mM Tris 7.5, 150 mM NaCl and 5 mM DTT, the solubility of the protein is decreases drastically and tend to crystallize while concentration. Protein cannot be concentrated more than 3 mg/mL, however I noticed white turbid protein if I force to concentrate 3mg/mL. When I observed this white turbid solution under the microscope, I noticed shower of tiny protein crystals which are needle in shape. I screened freshly purified protein (2.5 mg/mL) in different Hampton and Qiagen screens, strangely none of the conditions gave the crystals. I concentrated left over protein at 15oC at 3 mg/mL and kept in the 4oC for 4 days again I noticed shower of crystals. This protein solubility is increased to ~20mg/mL when I kept in 15 Tris 7.5, 400 mM NaCl, 5% Glycerol and 5 mM DTT, but did not crystallize while concentration and also after screening with Hampton and Qiagen screens. My queries are 1. How do I get the crystals in the crystallization set up rather than while concentration, so that I can control the diffusion and finally nucleation? 2. Could anybody give me suggestions on seeding in this type of situation? 3. Any comments on reverse vapor diffusion for this type of protein are most welcome. So I can keep protein in high ionic strength (~400 mM NaCl)and diffuse against low Ionic strength or deionized water? Or any other protocol? Any suggestions are well appreciated. Thanking you in advance Raj E. Rajakumara Postdoctoral
Re: [ccp4bb] domain contact surface
Hello Again, The buried surface area is twice the interface area, because the interface is counted twice when working out the solvent accessible area which disappeared upon interfacing, once on molecule 1 and once on molecule 2. This is a matter of definition. While I prefer to quote the interface area, quite a number of people prefer the buried surface area. Either one should be alright, as long as you say clearly which one you are reporting. Pierre Dr. Pierre Rizkallah, Senior Lecturer in Structural Biology, WHRI, School of Medicine, Cardiff University, Heath Park, CF14 4XN email: rizkall...@cf.ac.uk phone + 44 29 2074 2248 Jane Bailey jbailey.x...@googlemail.com 19/02/10 4:57 PM Ed Pozharski wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html Try PISA server - it will identify contacts and report total buried surface area per contact. On Fri, 2010-02-19 at 16:42 +0100, Jane Bailey wrote: Dear all, I am trying to calculate the domain-domain contact surface from one chain. I see AREAIMOL to only tell you the accessible surface area/residue. Could any other software/webserve could specify residues in the contact surface and calculate the total contact area? Thanks J. Dear all, Thanks very much for the answers. I managed to get the interface from PISA by assign two chain IDs onto two domains, as suggested by Pierre and Amy. It gave the interface Solvent-accessible area of each domain: 274.2, 312.2, but not the BSA. However I saw it gave the BSA of individual residue from the interface. Do people get the total interface BSA by adding them up? best Jane
Re: [ccp4bb] anomalous difference fourier maps
Dear Ivan, It is great to have confirmation that you got answers to your initial question that enabled you to solve your problem. The idea of the BB is that, in exchange for the gift of other people's contribution to solving your problem, you are expected to share the solution with the other subscribers. If you look through the archives, you will see numerous cases when people who received contributions, either as replies to the BB or in messages sent to them off-board, then took the trouble to collect them into a message to the BB as a whole, so that everyone learned something. It would be really good if you could do the same with the answers you got. I think I will bow out of this thread by retracting any churlish content in my previous posting. I felt it was not quite satisfactory that a question asked in terms of CCP4 programs should only leave a Phenix-based solution as a written trace - but avoiding this takes some work from the beneficiaries of the answers and not just a modicum of tact from the repliers in answering the questions that are actually being asked. Of course, any broadening of subject matter beyond the initial specific question asked is a wonderful thing to see happen in any scientific forum; but this was not at all the case in this particular instance. With best wishes, Gerard. -- On Fri, Feb 19, 2010 at 08:13:26AM -0800, xaravich ivan wrote: ok I think I should say something here.For some reason I was unable to find REPLY-ALL button and my reply did not go to everyone first so I had to write another message.I got the answer to my original query and I used CCP4 (CAD) and coot as suggested by Jan. Having said that I did not know that you could do it in so many ways. and that is perhaps the most valuable thing!!! Thanks all of you for your replies and I think the best thing of this forum is to be able to get answers when you do not have anyone in the lab to guide you. People like us need you guys the most. Irrespective of whether it is ccp4bb or phenix or any other program, the goal is to share the knowledge and help the less experienced (less fortunate) with your vast expertise, so that people can enjoy doing exciting crystallography related research. Thanks to everyone, Ivan On Fri, Feb 19, 2010 at 7:37 AM, Edward A. Berry ber...@upstate.edu wrote: The guidelines for the CCP4BB are extremely broad and certainly include discussion of other software packages. Since the original poster's question had to do with a specific problem with CCP4, it would have been appropriate for Pavel to prefix his reply with something like I hope you receive an answer to your question shortly, but in the meantime here is an alternative. But this is a nicety I would be glad to forgo for the sake of getting the extra information. The problem is not that the phenix team was so quick to promote their software, but rather that now 14 hours after the original post, no one has answered the CCP4 question. It is too easy to say yes, I think I could help this guy, but half the readers of this BB could probably give a better answer. Maybe someone at CCP4 should be assigned to answer all reasonable queries that go unanswered for more than 8 hours. The phenomenal rise in popularity of the phenix package is probably due as much to the incredible responsiveness of the phenix team, not only in support but in adding requested features, as it is to the power and ease of use of the programs. Vellieux Frederic wrote: Hi Dirk, When it happens that I reply to a ccp4bb message and that the answer, or solution I may have (which I think is better or more appropriate) involves using non-ccp4 programs, I do it off-list. By replying privately to the person who asked the question. Fred. Dirk Kostrewa wrote: Dear Gerard, I can only agree with you - I've also noticed a growing and sometimes irritating cross-advertisement of non-CCP4 programs on the CCP4BB over the last months (mainly Phenix). Unless, the specific task that was asked for, can only be (reasonably) solved with non-CCP4 programs, such replies leave a somewhat bad aftertaste. Personally, I think, it would be perfectly acceptable if both solutions with CCP4 programs and other programs would be given, so that the user may choose, or try them all. Best wishes, Dirk.
[ccp4bb] To calculate Domain-domain orientation
Dear all, Problem is coming one after another. I got another question about how to calculate the domain/domain orientations (tilt and twist angles). Sorry I probably disturb a bit often I searched a lot, but no publication says how it was calculated. Does anybody know anything about this? Thanks a lot! Jane
[ccp4bb] Fwd: [ccp4bb] anomalous difference fourier maps
Begin forwarded message: From: Charles W. Carter, Jr car...@med.unc.edu Date: February 19, 2010 10:28:55 AM EST To: George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de Subject: Re: [ccp4bb] anomalous difference fourier maps I'm also inclined to join this discussion. I agree with both Gérard and George. I also sympathize with Ivan. Over the years, I've used Bijvoet Difference Fouriers for many purposes, and every time I attempt to calculate one with CCP4 programs, I must spend an inordinate amount of time experimenting to find the commands that give the result I expect. Often I've kept a toy problem to ensure that I was doing it right. It has always been extremely aggravating, and several times I've had to resort to personal appeals to Eleanor to help me out. It should not have been so obscure, and if it remains so, then shame on CCP4! I also share Gérard's nostalgia for the days when virtually all crystallographers had first-hand experience with such questions. The enterprise has grown exponentially since then by the inclusion of increasing numbers of people from other fields who can take advantage of the institutionalization of computing software, pioneered by the CCP4, to extend structural biology into realms that were only dreams two decades ago. Both Gérard and George played pivotal roles in that transformation while I did not. Nonetheless, I sympathize with their explicit and implicit sentiments and remind others of two excellent papers describing this important method: Blow, DM (2003) How Bijvoet Made the Difference. Methods in Enzymology 374, Chapter 1. 3-22. Roach, Jeffrey (2003) The Bijvoet Difference Fourier Synthesis. Methods in Enzymology 374, Chapter 6. 137-145. Charlie On Feb 19, 2010, at 9:57 AM, George M. Sheldrick wrote: I am inclined to agree with Gerard. Of course if there is a specific question to CCP4bb about SHELX, I try to answer it. Since I am too lazy to maintain my own bb, this is very convenient. However I have stopped 'poaching', for example for the thread in question I resisted the temptation to point out that SHELXE has a convenient way of making such anomalous maps, because that would not have been a direct answer to the question and CCP4 provides similar facilities (and SHARP would be even better). We use the CCP4 programs (especially REFMAC and COOT) extensively and are very grateful for all the support we get. In particular, we should not underestimate the unique role CCP4bb plays in crystallographic education. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Fri, 19 Feb 2010, Gerard Bricogne wrote: Dear all, This is a remark I have wanted to make for a long time but managed so far to repress. However, this case is absolutely clear: Ivan was not asking for general advice on how to carry out a general task, but how to perform a specific task with the CCP4 software. In response we get (surprise, surprise, ...) another instance of the relentless touting for Phenix on the CCP4BB, which has long been an expected (or tolerated?) feature of this BB. Contributions from Phenix developers are of course much appreciated when questions are about general crystallographic matters where their expertise and experience is valuable; but when people ask specifically how to do something with CCP4 programs, could they please not be grabbed by the sleeve and enticed to buy their sweets from the shop next door? In this case, for instance, Ivan thanks guys (plural) for the answers he got (All of your suggestions were great). Perhaps one of those was a CCP4-based answer, but if so it has not even been communicated to the rest of the CCP4BB subscribers - so not only is this touting in bad taste after a while: it even interferes with the sharing of expertise in using the CCP4 software, which after all must be one of the main missions of this BB. I have long wondered whether anyone on the CCP4 side been assigned the task of answering every question to the Phenix BB by describing how to do it with CCP4 programs ... . With best wishes, Gerard. -- On Thu, Feb 18, 2010 at 03:53:02PM -0800, Pavel Afonine wrote: Hi Ivan, two ways (at least) to do it in PHENIX: - phenix.refine always computes anomalous difference Fourier map (provided that your input data file contains Fobs(+) and Fobs(-)). The command below will do it: phenix.refine model.pdb data.mtz strategy=none main.number_of_macro_cycles=0 output.prefix=maps_only - you can use phenix.maps that is a general tool to compute a broad variety of maps. Type phenix.maps from the command line for usage instructions. You need to have the latest development (or one of) PHENIX nightly build for this. All this is available from the GUI too. Pavel. On 2/18/10 3:34 PM,
[ccp4bb] ACA 2010, structural enzymology - mechanistic: call for abstracts
American Crystallographic Association, Inc 2010 Annual Meeting Chicago, IL July 24-29, 2010 http://www.amercrystalassn.org/content/pages/2010-meeting CALL FOR PAPERS Abstract Submission Deadline - March 31, 2010 Session Name: 01.05 Structural Enzymology: Mechanistic (BioMac) Day: Thursday morning July 29, 2010 Session Chair: Allen M. Orville, Brookhaven National Lab Macromolecular crystal structures of ligand complexes or reactive intermediates provide valuable mechanistic insight, but for which the crystallographers often find themselves interpreting mystery density within the data. Thus, the talks and posters in this session are intended to highlight crystal structures and the use of techniques that provide strong correlation(s) to the proposed reaction mechanism. This will likely include important correlations with complementary kinetic and/or spectroscopic techniques. Taken together, the multidisciplinary data provides compelling insights into the proposed reaction mechanism that an individual method has difficulty supporting in isolation. Three invited speakers will be selected from the abstracts submitted for the session. They will complement the three confirmed speakers: Dr. Brian G. Fox, University of Wisconsin, Madison, WI, will present new insights into the reaction cycle of diiron hydroxylase-effector protein complexes. Dr. George Richter-Addo, University of Oklahoma, Norman, OK, will discuss recent work on the photochemistry of heme-based reactions with single-crystal spectroscopy and x-ray diffraction. Dr. Paul R. Carey, Case Western Reserve University, Cleveland, OH, is a pioneer in single-crystal Raman microspectroscopy with applications in RNA and enzyme-based catalysis. Sincerely, AMO ** Allen M. Orville, Ph.D. Biology Department Brookhaven National Laboratory Upton, NY 11973-5000 e-mail: am...@bnl.gov phone 631-344-4739 fax 631-344-2741 http://www.bnl.gov/biology/People/Orville.asp
[ccp4bb] coot, atom connectivity
I have another basic question about the definition of the connectivity in residues. The side chains of my Se-methionines show up as disconnected atoms. There is no bond between the Se and the neighboring C atoms. Which library or data files contain the information about bond distances etc? Or how do I approach this best? Ursula
Re: [ccp4bb] how to keep chastity of CCP4BB [was: Re: anomalous difference fourier maps, or how to keep chastity of CCP4BB]
The current remit of CCP4BB is protein crystallography. As such, contributions from Phenix developers are welcome. Having said that, if the question is about how to perform some operation(s) with CCP4 then they perhaps ought to be somewhat circumspect in their response. If after a week or so the question has not been answered then a Phenix or other-non-CCP4-based answer would not be inappropriate (in my opinion). On to a different but related topic: I think CCP4 mailing list archive is poor place to store technical answers. I wonder if the repeated questions is because previous answers are so hard to find? And also the reason why many would rather answer in person rather than to the list? (I know that I answer personally more than I do to the list). I'd like to encourage the use of MajorGroove.org for many of the questions that come up on CCP4BB - it is (or will be) a great format/forum for questions and answers with a useful search and rating system. It will become better with more use. (MajorGroove.org is not the place for discussion.) Paul.
Re: [ccp4bb] anomalous difference fourier maps
Hi Everyone, I hope everyone gets this email. Below are the two answers I got on how to solve my problem using ccp4.I actually emailed another person who wanted to know how I did it. So I got to transfer what I learned immediately. But his email was offline not through the forum. The answers to my queries on how to make an anomalous difference fourier map using CCP4. The first answer is the one that I used, but I am attaching all the answers that I got. 1) Hi Ivan, coot can do that quite nicely. Make sure you have the DANOs in the same file as the phases, else use CAD to merge files. Then 'load mtz', go to 'Expert mode', select DANO as amplitudes, PHWT as phases, unselect 'difference map' and contour at somewhere between 3-5 sigma. 2) Xaravich, Just to clarify you are using: PHI=PHIC (SFALL), DANO=DANO (data), Sigma=SIGDANO (data) and WEIGHT=FOM (SFALL)..to my recollection, you shouldn't be using FCalc.. I've never used SFALL to generate phases. I would use the PHIC from the output mtz of refmac..but I think this is the same thing. All the other steps look correct to me. thanks, Ivan
