Re: [ccp4bb] an over refined structure
Hi Sun, On Mon, Feb 04, 2008 at 02:15:05PM -0800, Sun Tang wrote: I used NCS before rigid body refinement. After that I did not put NCS restraints in the restrained refinement and TLS+restrained refinement because it raised the R/Rfree quite a lot. Use NCS. Really! There is never a reason for switching off NCS restraints (ok _maybe_ at real atomic or ultra-high resolution ...). Obviously, you'll need to change the way you apply NCS restraints: from a simple per-chain definition to maybe a per-domain definition, taking out residues in crystal contacts, allowing for a different base B-factor of different chains/domains etc. Some programs do these things fairly automatically for you. This might make it awkward to use NCS sometimes, but at 2.8A it is a must (I think). And if your use of NCS increases Rfree, then there is aproblem in the setup of NCS restraints, not in the principal usage. Note: re-introducing NCS restraints might increase the R, but if the Rfree stays similar: who cares? -- See also: G J Kleywegt, Use of non-crystallographic symmetry in protein structure refinement, Acta Crystallographica, D52, 842-857 (1996). According to http://xray.bmc.uu.se/~gerard/citation.html a classic! paper [gosh ... I really like Gerard's style ;-)]. Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Question about strange MR solution
Dear Michele, I think your MR solution is on a different allowed origin - that's all Ciao Pietro -- Pietro Roversi Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3ER, England UK Tel. 0044-1865-275385
Re: [ccp4bb] an over refined structure
I agree that the difference in Rwork to Rfree is quite acceptable at your resolution. You cannot/ should not use Rfactors as a criteria for structure correctness. As Ian points out - choosing a different Rfree set of reflections can change Rfree a good deal. certain NCS operators can relate reflections exactly making it hard to get a truly independent Free R set, and there are other reasons to make it a blunt edged tool. The map is the best validator - are there blobs still not fitted? (maybe side chains you have placed wrongly..) Are there many positive or negative peaks in the difference map? How well does the NCS match the 2 molecules? etc etc. Eleanor George M. Sheldrick wrote: Dear Sun, If we take Ian's formula for the ratio of R(free) to R(work) from his paper Acta D56 (2000) 442-450 and make some reasonable approximations, we can reformulate it as: R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s) where s is the fractional solvent content, d is the resolution, p is the effective number of parameters refined per atom after allowing for the restraints applied, d^3 means d cubed and sqrt means square root. The difficult number to estimate is p. It would be 4 for an isotropic refinement without any restraints. I guess that p=1.5 might be an appropriate value for a typical protein refinement (giving an R-factor ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R-factor ratio of 0.277/0.215 = 1.29 is well within the allowed range! However it should be added that this formula is almost a self-fulfilling prophesy. If we relax the geometric restraints we increase p, which then leads to a larger 'allowed' R-factor ratio! Best wishes, 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. +49-551-39-2582
Re: [ccp4bb] Question about strange MR solution
I think you have just found a symmetry equivalent of your original structure solution. In P6122 there are 12 possible symmetry operators to choose from - only one of which will be the identity (equivalent to alpha=beta=gamma=0) One certainly will have alpha or gamma = 180, and beta 0, equivalent to -x,-y,z Then you could also have found a solution on the alternate origin (0,0,0.5) I use lsqkab ( superpose molecule task) to fit the solution to the original and look at the matrix to convert 1 to the other It should match one of the symmetry operators of P6122 Eleanor Michele Lunelli wrote: Dear all, I refined a protein structure in the space group P6(1)22, with one copy in the asymmetric unit, resolution ~1.8 A, Rwork=0.20, Rfree=0.22. Then I tried to feed Phaser (version 1.3.3) with this structure. It found quickly a very prominent solution, but the first euler angle is 180 instead of 0 degrees (the others are 0, as well as the fractional coordinates). This solution is not symmetry-related with the structure that I used as search model: indeed, there are a lot of clashes. However, when I refine this solution, I obtain immediately R factors as good as the search model, and also the electron density map looks perfect. Of course, I used the same reflections file to refine the initial structure and the MR solution rotated of 180 degrees. How can I explain this? The analysis with Truncate (moments and cumulative intensity distribution) don't suggest any twinning, as well as the Padilla-Yeates test. Is it possible, that I refined the structure in the wrong space group? Thank you in advance, Michele Lunelli
[ccp4bb] Call for applications to the Membrane Protein Laboratory at Diamond
Dear Crystallographers, First call for proposals to visit the Membrane Protein Laboratory (MPL) at the Diamond Light Source, Oxfordshire. The MPL is a facility for purification, crystallisation and structural studies of membrane proteins. It is located at the Diamond Light Source and is a collaborations with Imperial College London. The Director is Prof. So Iwata. The MPL is funded by the Wellcome Trust. This facility is open to applications from groups anywhere in the world. If you are interested in working at the MPL, please follow this link to read more: http://www.diamond.ac.uk/MPL/default.htm If you would like to apply, please download the forms from this page and return them to Liz Carpenter by the 27th of February. http://www.diamond.ac.uk/MPL/MPLUser.htm If you would like to discuss an application, please contact me by email: [EMAIL PROTECTED] Best wishes, Liz
[ccp4bb] Membrane Protein Workshop at Diamond 1st-3rd April 2008
Dear Colleagues, We are organizing a workshop on the 1st to the 3rd of April, 2008 Title: Workshop on Membrane Protein Crystallization and Crystallography Location: Diamond Light Source, Didcot, Oxfordshire, UK. Dates: 1st - 3rd April 2008 Funded by: E-MEP and EMBN-Train For more information please see the flier: http://www.diamond.ac.uk/CMSWeb/Downloads/diamond/MPL/ Membrane_Protein_Course_April_2008.pdf And the webpage: http://www.diamond.ac.uk/MPL/MPLuser.htm Best wishes, Liz Carpenter __ Dr Liz Carpenter, Membrane Protein Laboratory Group Leader, Diamond Light Source, Chilton, Didcot, Oxfordshire, OX11 0QX, UK. Tel: 01235 778517 [EMAIL PROTECTED] DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV
Re: [ccp4bb] an over refined structure
Hi Sun Your bond length angle RMSD's look suspiciously high for a 2.8 Ang structure; this usually means that some weighting parameter(s) is/are not optimal. 2.8 Ang is not that far from the point where the optimal choice of structure parameters may be torsion angles instead of Cartesian co-ordinates, in which case the optimal RMSD's for bond lengths angles would be exactly zero. You should be optimising all weights that have been set arbitrarily by the program (i.e. not obtained from independent experimental sources), this includes not just the X-ray weight but also the B-factor restraint weight(s) (the usual culprit) and the NCS restraint weight(s), as Clemens suggests. I now use the free log(likelihood) to optimise the weights rather than Rfree, this is now printed by newer versions of Refmac, but it's up to you which you believe (the difference in the results may not be significant anyway). Alternatively CNS phenix.refine have scripts which automatically optimise the weights (against Rfree) for you (maybe one day CCP4/Refmac will have this very useful capability ;-) ). Also I would check your waters manually - don't believe everything the auto-water placing software tells you, i.e. does the density look sensible (at least roughly spherical shaped), is it possible they are something other than water (check for excess density and/or suspiciously low B factor), do they all H-bond to protein and/or other waters you are confident about. I had a structure at 2.9 Ang where I found only 10 good waters and that was for 900 residues in the a.u. (maybe it had something to do with the fact that the solvent content and average B were quite high and the data was partially twinned so the map quality was poor). I'm sure others have opinions on how many waters you expect to find at various resolutions. HTH -- Ian -Original Message- From: Sun Tang [mailto:[EMAIL PROTECTED] Sent: 04 February 2008 22:32 To: Ian Tickle Cc: CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] an over refined structure Hi Ian, Thank you very much for your detailed information. I checked the effect of weighter term (wa) in CCP4i for the R/Rfree. When I used wa=0.01 , the value is 0.225/0.277 FOM =0.799. The values changed to 0.204/0.269 (FOM=0.806) for wa= 0.05, 0.195/0.268 (FOM=0.807) for wa=0.1 and 0.186/0.267 (FOM=0.807) for wa=0.2, respectively. It seemed that increase in wa decreases both R and Rfree with R more than Rfree. Which wa value is the best one in this case? Thank you very much for your valuable help. Best, Sun Ian Tickle [EMAIL PROTECTED] wrote: Hi Sun Tang Unfortunately there's no such thing as a fixed value for the maximum acceptable Rfree-Rwork difference that applies in all circumstances, because the 'normal' difference depends on a number of factors, mainly the observation/parameter ratio, which depends in turn on the resolution and the solvent content (a greater solvent content means a bigger cell volume which means more reflections for a given number of ordered atoms in the a.u. and hence a bigger obs/param ratio). The Rfree-Rwork difference also depends on Rwork itself (i.e. you tend to get higher values of Rfree-Rwork for higher values of Rwork), so it's better to think in terms of the Rfree/Rwork ratio (which is independent of Rwork). So for example at very high resolution a 'normal' value for Rfree-Rwork might be only 0.02 (so 0.05 which is what many people consider acceptable would actually be unacceptably high), whereas at low resolution it might be 0.1 (so 0.05 would be unacceptably low). Also you need to bear in mind that Rfree tends to have a quite high uncertainty, particularly at low resolution (because it's usually based on a relatively small number of observations), so the deviation has to be quite big (e.g. 3 SU) before it can be considered to be statistically significant. So Rfree needs to be compared not with Rwork at all but with the value of the optimal Rfree/Rwork expected on the basis that the model parameterisation and weighting of X-ray terms and restraints are optimal and the errors in the model have the same effect as the random experimental errors in the data (i.e. a statistical 'null hypothesis'). As Tim just pointed out we tried to do this in our Acta D (1998) papers: there you can compare your observed Rfree/Rwork ratio either with the theoretical value or with the value found for 'typical' structures in the PDB at the same resolution. An abnormal Rfree/Rwork ratio could arise from a number of causes, not just over-fitting (I assume that's what you mean by 'over-refinement' - it's not clear to me how a structure can be 'over-refined' since a fundamental requirement of the maximum likelihood method is that the structure is always refined to convergence, and refining beyond that will by
[ccp4bb] Influenza M2 proton channel structures
Dear All, In the latest Nature (which for once arrived in a few days to our office...) there are interesting structures of the influenza M2 proton channel. One is by NMR, which resulted in a model of a closed state with four inhibitor molecules bound to the outside. Another is by X-ray crystallography, where an open state is observed. The x-ray structure is at 2 Angstrom without inhibitor, and they include a complex with the inhibitor at 3.5 Angstrom, where the inhibitor is modelled in positive difference density inside the pore. Drug-resistant mutations are near the binding site proposed in the X-ray paper, but the resolution is limited (3.5 Angstrom). The NMR paper also explains the drug- resistant mutations, but perhaps less convincingly. With the current evidence, which is more convincing? Or could both be true? Of course, we can always say we should wait for more evidence, like a higher-resolution X-ray structure or more biochemical data. In any case, I am curious to hear the opinions of other structural biologists. Carnaval greetings, Mark van Raaij
[ccp4bb] X-ray equipment donation
Available immediately for free from JJ in Springhouse, PA: Bruker AXS ProteumR system- Smart 6000 CCD area detector 6 kW rotating-anode generator Packed on shipping pallets, measures 83”x52”x72” For additional details please contact: Sean Moroney (JJ) email: [EMAIL PROTECTED] Please contact Sean directly. I have no additional information pertaining to this hardware other than what Sean gives me which is listed above. Would love to keep this gear out of the landfill. Best regards, Matthew B. Neiditch, Ph.D. Assistant Professor UMDNJ-NJMS Dept. of Microbiology and Molecular Genetics 225 Warren St., Room E450U Newark, NJ 07101 Ph (973)972-8980
[ccp4bb] Off Topic:Docking
Dear ccp4 Community, I am sorry for the off topic question. As you all aware there are many docking programs available from commercial vendors/. And everyone claims that their product is the best in the business. Here i would like to ask for some experiences from this wonderful community. I have few questions: 1. How do you choose which program to use for your protein. 2. Is it depends on your active site 3. Is there any literature on how to go about it Please help me in this regard.. Each program is giving different results and not even comparable.. Thanks for your inputs regards John
[ccp4bb] Alternative origin list in CCP4 documentation
Dear all In the CCP4 documentation about alternative origins for spacegroups (http://www.ccp4.ac.uk/dist/html/alternate_origins.html) one can find the following table: _ P 1 m 1 SG No: 6 (Standard short HM symbol: Pm) Number of alternate origins: 2 This is a polar spacegroup: the origin is not fixed along the C axis Norigin Xo Yo Zo 1 0. 0. ?? 2 0. 0.5000 ?? _ However, I would expect that the choice of the origin for this space group be arbitrary on the mirror plane, i.e.: _ The origin is not fixed in the AC plane Norigin Xo Yo Zo 1 ?? 0. ?? 2 ?? 0.5000 ?? _ Similarly, I would expect that the tables for space group P1c1 (No 7), C1m1 (No 8) be different from those in the documentation (I have not checked for other space groups ...) Is it correct what I am saying or am I missing something? Thank you --- Stefano TRAPANI Centre de Biochimie Structurale (CBS) CNRS UMR 5048; UM 1; UM 2; INSERM UMR 554 29 rue de Navacelles 34090 MONTPELLIER Cedex, France Tel : +33 (0)4 67 41 77 29 Fax : +33 (0)4 67 41 79 13
Re: [ccp4bb] Still cannot read .mtz + another ? part II
PJ. Ok after doing the stuff for refmac and running a few test runs of it, it worked ok. After using it on my current model, all of my bfactors shot up, my RMS for bond lengths went from .015 to 0.34 and my RMS for angles were also doubled. (this was done by re-running a job in refmac prior to my new changes) Thus I am changing things back to the way they were, in doing so I noticed another folder with a link to $CLIB/lib/data/monomers which is the setenv MOLREPLIB section just above the CLIBD_MON section would these need to be changed as well? Also still no word out there from you all about PHASER =) Thanks everyone, the advice on this board is fantastic Ainsley P.J.Briggs wrote: Dear Ainsley I'm not sure which file you took from Garib's page, however the CCP4i install options (under the System Administration menu) are only for installing new interfaces and not for updating the programs themselves. I just took a look at the files on Garib's page, and downloaded the file linked as refmac 5.4 for linux, which arrives on your system as refmac5.4_linintel.tar.gz. Unpacking this I get three files: refmac_linintel makecif_linintel libcheck_linintel These are updated executables for the refmac5, makecif and libcheck programs that should be in the bin directory of your CCP4 distribution (do cd $CBIN to get there) - you should move the current executables out of the way, and then copy and rename the new files into the bin directory. For the new dictionary, I would suggest downloading the file and unpacking it somewhere like the lib/data/ subdirectory of your CCP4 distribution. This should create something like /your/path/ccp4-6.0.2/lib/data/dic You can then change the environment variable CLIBD_MON to point to /your/path/ccp4-6.0.2/lib/data/dic/, either on the command line or in ccp4.setup (probably better to do the latter). I'm pretty sure that this should work - sorry if it sounds complicated. And yes it would probably be useful if Garib did indeed include some information about updating on the actual page - but we don't have any control over that. Hope that this helps, best wishes Peter Briggs CCP4 C.Ainsley Davis wrote: Hey all I have checked everything that was mentioned here. I dont have PHENIX installed ( I am using the current version from http://diablo/ucsc.edu/~wgscott/debian/deb/ccp4/) I checked the setup file and its set to 1 Any other ideas? Next question I would like to install the newest version of REFMAC. I downloaded the file from Garib's page) I try to use the ccp4 GUI to install the file, but it doesnt like it. It says something about it being unable to read the contents. If I gunzip then tar the file i get 3 files which I am not sure what to do with. There is also a new library file Garib recommends downloading, but in any case I cannot install the 5.4 version of REFMAC and cannot find any documentation online on how to install it! Thanks again CCP4 BB!
Re: [ccp4bb] xtalview and mifit
On Tuesday 05 February 2008 16:51, John Badger wrote: One suggestion on the XtalView/xfit problem is that it might be a result of trying to run on a 64-bit computer. No, that's not it. XtalView/Xfit runs just fine on 64-bit. The problem is that both Fedora and Suse 10.3 currently ship with a broken xorg library. This is a known problem (Google for details), but I do not know if there is a fixed version available for download. This particular xorg build error is not present in Mandriva's rpm for the same set of libraries; I can run Xfit just fine on both 32-bit and 64-bit systems with it. In principle one could perhaps re-install xorg from the Mandriva 2008 rpms to replace the one is Suse 10.3, or replace just the one library by hand, but I would hesitate to take that route unless I was willing to reinstall from scratch if it failed. -- Ethan A Merritt
Re: [ccp4bb] xtalview and mifit
This is my response to Marius from earlier today. I should have sent it to the list as well. This was done in Fedora 8, but it I'm guessing similar commands in the other distros that now use libxcb will also work. BTW, I tried upgrading to libxcb-1.1 and using the sloppy_lock variable that fixes some other programs with this error but it did not work for me. --paul I believe I have solved it, though it isn't too pretty. I essentially used the method people have used for nmrdraw: http://tech.groups.yahoo.com/group/nmrpipe/message/1630 This involves downgrading to the earlier packages (see the link) by doing something like this: rpm -U --force --oldpackage --nodeps libX11-1.0.3-8.fc7.i386.rpm libX11-devel-1.0.3-8.fc7.i386.rpm copy the library files from /usr/lib to the xtalview lib: cp /usr/lib/libX11.so.6.2.0 /usr/local/XtalView/lib/ibmpclinux2/ go to xtalview lib directory and make sym links: ln -s libX11.so.6.2.0 libX11.so ln -s libX11.so.6.2.0 libX11.so.6 and now do a 'yum update' to replace the libX11 packages. I just got this working and haven't tested it much. I just ran xfit and opened a model. I haven't tried anything else from xtalview. --paul John Badger wrote: A reference to Xtalview/xfit and MIFit came up in a thread without subject line. The poster is correct that MIFit is a successor to XtalView. MIFit is under active development and is free to academics. It runs on Windows and Linux. MIFit can be obtained from http://mi.active-sight.com/download.html Besides the model-fitting applications, MIFit contains a set of applications for structure solution and refinement as well as a ligand dictionary editor for generating ligand refinement restraints. One suggestion on the XtalView/xfit problem is that it might be a result of trying to run on a 64-bit computer.
