Re: [ccp4bb] MR problem --molrep
Hi there, I think 55% completeness is insufficient - you really need to collect more data. Take a look at this movie from James Holton's website: http://ucxray.berkeley.edu/~jamesh/movies/osc.mpeg Stop the movie at 55% and check out how lousy the maps are compared to 95-100% - and I think these maps are generated with perfect phases, at 1.5A resolution. Molecular replacement programs and refinement programs are going to struggle with just over half a dataset - this is also why your R-factors are sky high. In addition, I know of cases where reviewers have cast doubt over structures (of a point mutant) with data of 80% completeness. I don't think a 55% complete dataset is going to get out there. I really think more data is the only way our of your predicament. I hope you have a few crystals ready to go! Good Luck, David. 2008/7/27 Carl Soja [EMAIL PROTECTED]: HI all, Thank you very much for your kindly answer. I also tried to use different program like phaser, amore, and some MR servers. actualy, I don`t know how many molecule in AU clearly. 2-6 molecules in 76%- 35% of solvent content. 4mer is 50% of solvent content. the homolgous structure has high solvent content over 70%. when i used phaser program to do MR, I can get several solutions but not good structure conformation. I don`t know how to use phaser do automatic more than 2 molecules search like molrep program. As spacegroup, I checked the C222 and C2221. the phaser program can give both solutions when I changed the search model or maximum solution limit. my question is how i can search more than 2 molecules by phaser program like molrp only input over 2. by the way, my diffraction data have low completeness(55%) and low redandancy(1.8). Thanks in advance!! carl roja -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
[ccp4bb] Postdoctoral and Ph.D. positions in Milano
Postdoctoral research position and Ph.D. position in Structural Biology A postdoctoral research position and a Ph.D. position in structural biology are available in the newly established group of Marina Mapelli at the IFOM-IEO Campus in Milano (http://www.ifom-ieo-campus.it/research/mapelli.php). The main interest of the group focuses on the structural and functional aspects of protein complexes governing spindle orientation in asymmetrically dividing cells. Postdoctoral research position I am seeking a motivated and enthusiastic postdoctoral fellow with interests in structural biology and in mechanisms underlying protein functioning in cellular processes. Applicants should have recently obtained a Ph.D. in biochemistry or equivalent qualification in a relevant research area. Some experience in recombinant multi-protein complex reconstitution, enzymology or x-ray crystallography is desirable. Ph.D. position Ph.D. candidates should have terminated their university degree in Life Science disciplines (preferably Biochemistry, Biology, Chemistry). Some hands-on experience in biochemistry or structural biology would be advantageous, but not required. Willingness to study protein functions with biochemical and biophysical methods, as well as interest in learning protein crystallography is essential. The Ph.D. position is within the SEMM 'Ph.D. in Molecular Medicine' programme (http://www.semm.it/). The Structural Biology Department of the IFOM-IEO Campus is equipped with the state-of-the-art apparatus for protein purification and crystallization, including a nanodrop crystallization robot and an automated imaging system, and has good access to the synchrotron beamlines. Successful candidates will benefit from a stimulating and collaborative environment within the Campus (http://www.ifom-ieo-campus.it/). Postdoc applicants should send their enquiries by e-mail to Marina Mapelli ([EMAIL PROTECTED]). They should also ask two referees to send letters of recommendation at the same electronic address. Ph.D. candidates should apply to the SEMM Ph.D. school within September 21, 2008 (see guidelines at http://www.semm.it/appli_mm.php). -- - Marina Mapelli, PhD Department of Experimental Oncology European Institute of Oncology Via Adamello 16, I-20139 Milan, Italy tel: ++39-02-9437-5018/5042 fax: ++39-02-94375990 email: [EMAIL PROTECTED]
[ccp4bb] 6.0.99
Downloaded CCP4 6.0.99 core and phaser. Did gunzip and tar to extract. Next, the instructions say for the first 3 tarballs unpack into the same directory, then configure...make...make install. I did ./configure and got this: ! Please set environment variable BINSORT_SCR (see installation instructions). ! Please set environment variable CCP4_SCR (see installation instructions). ! Please set environment variable CINCL (see installation instructions). ! Please set environment variable CLIBD (see installation instructions). ! Please set environment variable CCP4_OPEN (see installation instructions). ! Please set environment variable PUBLIC_FONT84 (see installation instructions). So apparently a config file is necessary first. Oops. Full text of ./configure output is attached. - === With the single exception of Cornell, there is not a college in the United States where truth has ever been a welcome guest - R.G. Ingersoll === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University [EMAIL PROTECTED] # ./configure Please read the following NOTICE TO THE LICENSEE Please read the following Conditions of Use and the appropriate Licence Agreement carefully before completing the installation process and using the software. These Conditions and the Licence Agreement are legal documents, and together contain information on allowed usage of the software suite, warranty information and liability disclaimers. If you do not agree with these Conditions, then do not complete the installation and do not use the software. These conditions have been updated for release 6.0 of the CCP4 software suite, and all sites are required to agree to these conditions of use. You should only be prompted for this agreement once. The Conditions are as follows: a. For commercial users and those doing commercial work: Please contact CCP4 at [EMAIL PROTECTED] to arrange a commercial licence. If you have arranged or renewed a commercial licence in the last 12 months, then no action is required. By agreeing to these conditions, you confirm that you have a valid and current CCP4 commercial licence. b. For users at non-profit sites and not engaged in commercial work: Please print and complete an Academic licence, sign it and post or fax it to CCP4 at the address given on the licence (email is not acceptable). A new licence agreement is required for CCP4 version 6.0. This licence is valid for 6.0 and subsequent versions until further notice. Copies of the licence can be found in a variety of formats in the top directory of the CCP4 distribution (files academic_software_licence.*) and on the CCP4 web site at www.ccp4.ac.uk/ccp4license.html . By agreeing to these conditions, you confirm that you have returned a licence for CCP4 version 6.0 . It is sufficient to send the licence - there will be no confirmation that it has been received. I agree to the above conditions and have done the actions required. y/n [n] ?:y Thank you. The CCP4 configure will now run as normal. srcdir bindir libdir /usr/local/ccp4_master/ccp4-6.0.99e /usr/local/ccp4_master/ccp4-6.0.99e/bin /usr/local/ccp4_master/ccp4-6.0.99e/lib OK, setting up for a system type of `linux'... ! Using gnu fortran compiler. If you have an old fortran compiler, you may be better ! off using f2c, see --with-f2c option. Will attempt to install the xwindows programs from `x-windows'. This is not supported for all systems. please note the xwindows programs will be compiled with an option that means errors are ignored. This could result in some xwindows programs not being compiled. If you want to disable the xwindows programs use --disable-x The CCIF library will be built and installed. This has been tested on the most common systems, but if you have problems, use the --disable-ccif flag. The CCTBX library will be built and installed. If you have problems, use the --disable-cctbx flag. Phaser will be built and installed. If you have problems, use the --disable-phaser flag. The LAPACK linear algebra package will be included in the installation. Configure will search for existing LAPACK and BLAS libraries on on your system - if a version of LAPACK is not found then the NetLib version will be built for you automatically. If you want NetLib LAPACK and BLAS to be used regardless of system libraries then rerun configure using --with-netlib-lapack To disable inclusion of LAPACK, use the --disable-lapack flag (This is not recommended as some programs will fail to build) Configure will try to
Re: [ccp4bb] 6.0.99
You need to source the include/ccp4-setup.whateveryourshellis file (after you edit it) in order to set up the environment. I started playing with the files to make them more automatic, if you want to try: http://sage.ucsc.edu/~wgscott/setup/ The one for zsh works the best, followed by bash (if you are using = bash 3.0). Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Jul 27, 2008, at 12:07 PM, David J. Schuller wrote: Downloaded CCP4 6.0.99 core and phaser. Did gunzip and tar to extract. Next, the instructions say for the first 3 tarballs unpack into the same directory, then configure...make...make install. I did ./configure and got this: ! Please set environment variable BINSORT_SCR (see installation instructions). ! Please set environment variable CCP4_SCR (see installation instructions). ! Please set environment variable CINCL (see installation instructions). ! Please set environment variable CLIBD (see installation instructions). ! Please set environment variable CCP4_OPEN (see installation instructions). ! Please set environment variable PUBLIC_FONT84 (see installation instructions). So apparently a config file is necessary first. Oops. Full text of ./configure output is attached. - = == With the single exception of Cornell, there is not a college in the United States where truth has ever been a welcome guest - R.G. Ingersoll = == David J. Schuller modern man in a post-modern world MacCHESS, Cornell University [EMAIL PROTECTED] junk.juhk
Re: [ccp4bb] Meaning of sigma level of electron density map?
Hi, P K, I think of it in terms of the probability of finding an electron within the volume enclosed by the electron density map. In general, the probability of finding an electron close to the nucleus will be higher than that of finding an electron far from the nucleus due to electrostatic attraction between the nucleus and the electron. If you're talking about a Gaussian distribution, the 1sigma, 2sigma, and 3sigma levels correspond, respectively, to 68%, 95%, and 99.7% of the population. As you've observed, an electron density map contoured at 3sigma is very tightly contoured around the nuclei. This means that there's a very high probability of finding electrons within the volume of the electron density map, i.e. close to the nuclei. The same electron density map contoured at 1sigma will be loosely contoured around the nuclei since there's less probability of finding electrons farther away from the nuclei. Good luck, Hidong P K [EMAIL PROTECTED] Sent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK 07/25/2008 12:38 PM Please respond to P K [EMAIL PROTECTED] To CCP4BB@JISCMAIL.AC.UK cc Subject [ccp4bb] Meaning of sigma level of electron density map? I am new to the filed of crystallography. I am having trouble figuring out what exactly does sigma level of electron density map mean. When sigma level of a map is increased (say from 1.5 sigma to 2 sigma) why the map covering individual residues becomes less wide and more precise? Shouldn't it be the other way round if they have anything to do with the Gaussian districution? Thanks!
Re: [ccp4bb] question about getting rid of model bias in refinement
Hello Charlie, Thank you very much for your comments. I mostly agree with you. However, as far as I know most of the complexes structures are solved with MR with the their apo-enzyme as search model and refined the structures with CCP4 or CNS. I tried the simulated annealing omitting the residue and 4 neighboring residues on each side and I found the conformation are essentially the same. I also tried to use composite omit-map calculation in CSN but I gave it because it took several days of computer time but only finished only 1/4 of the calculation. I understand the starting from the beginning is one choice. I wonder whether there are other easier ways in CCP4 to deal with this situation because this problem is quite common in refinement. I appreciate all the replies to my questions and I say Thank you very much here. Best, Sun --- On Sat, 7/26/08, Charles W. Carter Jr. [EMAIL PROTECTED] wrote: From: Charles W. Carter Jr. [EMAIL PROTECTED] Subject: Re: [ccp4bb] question about getting rid of model bias in refinement To: CCP4BB@JISCMAIL.AC.UK Date: Saturday, July 26, 2008, 3:15 PM Sun, I'm most of the way to one side of this debate: I believe that it is not possible to emerge fully from model bias without avoiding it in the first place with experimental phases. I may be overly pessimistic, but have considerable experience supporting at least skepticism. My interpretation of the experimental result you describe is that the covariances among the parts of the structure you left in place and those side chains you omitted is so strong and extensive that you'll never see the correct density coming back upon refinement, because other parts of the structure are ever so slightly off their true mean positions to compensate for the (evidently false) positions of the residues you omitted. Bill's suggestion that you actually refine the structure using simulated annealing without the omitted residues is an improvement over what you did, but it will require many cycles to get a much better approximation, and there is really no way to be sure when you can be confident. Starting the entire refinement over is a more aggressive strategy. If you decide to try this, you should examine the projection of the residue by residue real-space correlation coefficients across the entire sequence to ensure that you have only one population of values and delete all residues that comprise any population that has a distinctly different real-space correlation coefficient, building them back into the structure as it refines. That is, you should ensure that you don't begin refining any residues at the very beginning for which there is evidence that they might be different from their positions in your molecular replacement model. Charlie On Jul 26, 2008, at 2:12 PM, William G. Scott wrote: Hi Sun: It might be worth doing a simulated annealing omit refinement in phenix or CNS, with the residues in question omitted. CNS also allows you to make a composite-omit map. I haven't seen that in phenix yet but presumably it is doable. Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Jul 25, 2008, at 10:53 PM, Sun Tang wrote: Hello Everyone, I have a question about getting rid of model bias in refinement with refmac. I solved the structure with molecular replacement. After final refinement of the structure, I found out some key amino acids in the structure and wanted to make sure their conformations are correct. I omitted these amino acids (by setting occupancy to zero) and refined the structure. I manually fit the amino acids into the density and refined the structure again. I found these amino acids return to the precious conformations even though the conformations I fit were different. Should I omit these amino acids from the beginning of the refinement? What is the best way to get rid of the model bias? Your suggestions are greatly appreciated! Best, Sun **UNCrystallographers NOTE new website url** [EMAIL PROTECTED] http://xtal.med.unc.edu/CARTER/Welcome.html Department of Biochemistry and Biophysics CB 7260 UNC Chapel Hill, Chapel Hill, NC 27599-7260 Tel: 919 966-3263 FAX 919 966-2852
Re: [ccp4bb] question about getting rid of model bias in refinement
Dear Sun: Starting the refinement again, with a molecular replacement structure in which the residues in question are deleted, shouldn't take that much time. However, I would be really surprised to see much model bias in a sigma-A weighted 2Fo-Fc map generated after an omit simulated annealing procedure, especially if you crank the temperature up to 6000K. Another possible approach is to use EDEN. I put it on googlecode: http://code.google.com/p/edencrystallography/ I recently solved an RNA structure just with molecular replacement of random A-helix fragments. With this sort of psychotic approach to molecular replacement, model bias is a major problem. One trick that helped was to blur the HL coefficients (there is a script to do so in CNS) and then solvent-flatten/flip. But if the first approach isn't working, this is telling you something I think. Is it possible the result you are getting is correct after all? William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Jul 27, 2008, at 6:19 PM, Sun Tang wrote: Hello Charlie, Thank you very much for your comments. I mostly agree with you. However, as far as I know most of the complexes structures are solved with MR with the their apo-enzyme as search model and refined the structures with CCP4 or CNS. I tried the simulated annealing omitting the residue and 4 neighboring residues on each side and I found the conformation are essentially the same. I also tried to use composite omit-map calculation in CSN but I gave it because it took several days of computer time but only finished only 1/4 of the calculation. I understand the starting from the beginning is one choice. I wonder whether there are other easier ways in CCP4 to deal with this situation because this problem is quite common in refinement. I appreciate all the replies to my questions and I say Thank you very much here. Best, Sun --- On Sat, 7/26/08, Charles W. Carter Jr. [EMAIL PROTECTED] wrote: From: Charles W. Carter Jr. [EMAIL PROTECTED] Subject: Re: [ccp4bb] question about getting rid of model bias in refinement To: CCP4BB@JISCMAIL.AC.UK Date: Saturday, July 26, 2008, 3:15 PM Sun, I'm most of the way to one side of this debate: I believe that it is not possible to emerge fully from model bias without avoiding it in the first place with experimental phases. I may be overly pessimistic, but have considerable experience supporting at least skepticism. My interpretation of the experimental result you describe is that the covariances among the parts of the structure you left in place and those side chains you omitted is so strong and extensive that you'll never see the correct density coming back upon refinement, because other parts of the structure are ever so slightly off their true mean positions to compensate for the (evidently false) positions of the residues you omitted. Bill's suggestion that you actually refine the structure using simulated annealing without the omitted residues is an improvement over what you did, but it will require many cycles to get a much better approximation, and there is really no way to be sure when you can be confident. Starting the entire refinement over is a more aggressive strategy. If you decide to try this, you should examine the projection of the residue by residue real-space correlation coefficients across the entire sequence to ensure that you have only one population of values and delete all residues that comprise any population that has a distinctly different real-space correlation coefficient, building them back into the structure as it refines. That is, you should ensure that you don't begin refining any residues at the very beginning for which there is evidence that they might be different from their positions in your molecular replacement model. Charlie On Jul 26, 2008, at 2:12 PM, William G. Scott wrote: Hi Sun: It might be worth doing a simulated annealing omit refinement in phenix or CNS, with the residues in question omitted. CNS also allows you to make a composite-omit map. I haven't seen that in phenix yet but presumably it is doable. Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Jul 25, 2008, at 10:53 PM, Sun Tang wrote: Hello Everyone, I have a question about getting rid of model bias in refinement with refmac. I solved the structure with molecular replacement. After final refinement of the structure, I found out some key amino acids in the structure and wanted to make sure their conformations are correct. I omitted these amino acids (by setting occupancy to zero) and refined the structure. I manually fit the amino acids into the density and refined the structure again. I found these amino acids return to the precious conformations even though the conformations I fit were different. Should I omit these amino acids from
Re: [ccp4bb] question about getting rid of model bias in refinement
Hi Sun, you may also want to have a look at this paper: Acta Cryst. (2008). D64, 515-524. Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias. Pavel. On 7/25/2008 10:53 PM, Sun Tang wrote: Hello Everyone, I have a question about getting rid of model bias in refinement with refmac. I solved the structure with molecular replacement. After final refinement of the structure, I found out some key amino acids in the structure and wanted to make sure their conformations are correct. I omitted these amino acids (by setting occupancy to zero) and refined the structure. I manually fit the amino acids into the density and refined the structure again. I found these amino acids return to the precious conformations even though the conformations I fit were different. Should I omit these amino acids from the beginning of the refinement? What is the best way to get rid of the model bias? Your suggestions are greatly appreciated! Best, Sun
