Re: [ccp4bb] Only refine Bs in Refmac?
It should be on the latest ccp4. But as I understand ccp4 people extremely busy to make the latest version of ccp4 available. Regards Garib On 5 Sep 2013, at 10:36, Eleanor Dodson wrote: What has happened to the CCP4 update procedure? Shouldnt this mean latest versions are on that web site? e On 4 September 2013 22:57, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: Hi refine bref bonly should be what you are looking for. You may need to use the latest available version (5.8) from our LMB site: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/ With best regards Garib On 4 Sep 2013, at 19:59, hari jayaram wrote: Hi, How does one only refine Bs in refmac without changing the model coordinates . Is this accomplished using a zero cycle refinement with b-refinement set. I have never had to do this till now and didnt know how to set it up. Thanks Hari Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/ Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Only refine Bs in Refmac?
Hi Bill, I will add it tomorrow. However with new ccp4 release source code update should be straightforward (I hope). Regards Garib On 5 Sep 2013, at 15:15, William G. Scott wrote: On Sep 4, 2013, at 2:57 PM, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: You may need to use the latest available version (5.8) from our LMB site Hi Garib: Would it be possible to add a link for the source code, so this could also be used with Coot? Thanks. Bill Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Only refine Bs in Refmac?
You do not need anisoonly. You need bref only. If input pdb has aniso card then refmac will assume that mixed refinement should be done. If you want to add aniso for some of the atoms then you can use instructions like: brefine mixed aniso residue 100 A atoms FE S C* Please have a look documentation from the our LMB site for further details. Regards Garib On 5 Sep 2013, at 15:50, hari jayaram wrote: Thanks Garib for the new version. For only an anisotropic B refinement ..what are the keywords. The one below seems to work refi - type REST - resi MLKF - meth CGMAT - bref anisoonly ncyc 5 As far as Bill Scotts question does coot not pick up refmac5 and libcheck from $CCP4_BIN ? Thanks Hari On Thu, Sep 5, 2013 at 10:15 AM, William G. Scott wgsc...@ucsc.edu wrote: On Sep 4, 2013, at 2:57 PM, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: You may need to use the latest available version (5.8) from our LMB site Hi Garib: Would it be possible to add a link for the source code, so this could also be used with Coot? Thanks. Bill Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Only refine Bs in Refmac?
Hi Hari Only keyword you need (Assuming that you have aniso in your pdb) refinement brefinement only Did you try the version (5.8 version) from our MRC-LMB wbesite. In older version refinement brefinement only would do isotropic refinement only. I think I have changed and to make sure that it works for mixed and aniso also. If 5.8 version does not do what you want please let me kno. regards Garib On 5 Sep 2013, at 17:06, hari jayaram wrote: Hi Garib, Thanks for your help. I read the document at this location. Without mixed keyword the default iso B's kick in, so this statement below did not work. I took in a pdb with ANISO B's and ran a B-factor refinement and outputs a PDB without ANISOU records for every atom # DId not work when I had anisou records for all atoms in input refine- bref bonly This works and does the full anisotropic only refinement refine- bref aniso bonly This also works ( I was just guessing before I saw your previoud email) but It probably is equivalent to the one above. refine- bref anisoonly Thanks for your help Hari On Thu, Sep 5, 2013 at 11:24 AM, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: You do not need anisoonly. You need bref only. If input pdb has aniso card then refmac will assume that mixed refinement should be done. If you want to add aniso for some of the atoms then you can use instructions like: brefine mixed aniso residue 100 A atoms FE S C* Please have a look documentation from the our LMB site for further details. Regards Garib On 5 Sep 2013, at 15:50, hari jayaram wrote: Thanks Garib for the new version. For only an anisotropic B refinement ..what are the keywords. The one below seems to work refi - type REST - resi MLKF - meth CGMAT - bref anisoonly ncyc 5 As far as Bill Scotts question does coot not pick up refmac5 and libcheck from $CCP4_BIN ? Thanks Hari On Thu, Sep 5, 2013 at 10:15 AM, William G. Scott wgsc...@ucsc.edu wrote: On Sep 4, 2013, at 2:57 PM, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: You may need to use the latest available version (5.8) from our LMB site Hi Garib: Would it be possible to add a link for the source code, so this could also be used with Coot? Thanks. Bill Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/ Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Only refine Bs in Refmac?
Hi refine bref bonly should be what you are looking for. You may need to use the latest available version (5.8) from our LMB site: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/ With best regards Garib On 4 Sep 2013, at 19:59, hari jayaram wrote: Hi, How does one only refine Bs in refmac without changing the model coordinates . Is this accomplished using a zero cycle refinement with b-refinement set. I have never had to do this till now and didnt know how to set it up. Thanks Hari Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Overide refmac restraints
Hi Joel IF you use external restraints then you need to add type 0 at the end. Then refmac will assume that you want to override standard restraints. I.e. exte angle first chain A resi 1 atom S12 second chain A resi 1 atom C10 third chain A resi 2 atom N value 120 sigma 3.0 type 0 It is very strange that refmac is overriding definitions from your cif file. Would it be possible to have a look your cif file and how use it in refmac. For example some portion of your protein with cif file might be helpful. Regards Garib On 13 May 2013, at 22:45, Joel Tyndall wrote: H ithere, I am trying to refine a covalently bound ligand to my protein and I am having trouble with the restraints. I have generated the cif file and link within Jligand and this is reasonable. However it appears that REFMAC is overriding these and fitting the ligand to the density. I have added a keyword text file with externtal restraints such as : exte angle first chain A resi 1 atom S12 second chain A resi 1 atom C10 third chain A resi 2 atom N value 120 sigma 3.0 The resulting structures has incorrect angles which do not match the external restraints or cif file. Am I missing something? Any help muchly appreciated. (I am using the most upto date version of the CCP4 package on a PC) Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Puzzling Structure
It is typo: R factor for p212121 - 0.4 for p21212- around 0.18 Although water seem to have been moved around using p212121 On 12 Apr 2013, at 16:33, Phoebe A. Rice wrote: Looks like a typo to me: if you change the CRYST space group record from P212121 to P21212, as the paper says it is, the packing problem goes away. ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel Fodje [michel.fo...@lightsource.ca] Sent: Friday, April 12, 2013 2:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling Structure By the way, you will need to show symmetry atoms to see the problem. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Michel Fodje Sent: April-12-13 1:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Puzzling Structure Has anyone else noticed a problem with the structure of the N-terminal capsid domain of HIV-2 PDB 2wlv. Load it up to in coot and navigate to residue B118. /Michel. Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04
Dear all I think this error has been dealt with (Ed will correct me if I am wrong). The problem was -static in compilation. For whatever reason in some gcc (gfortran) -static does not work (it compiles but has problems in running, what is the reason is not clear to me). Sometimes in later gcc -static-libgcc -static-libgfortran works but not always. These flags are needed for distribution purposes. If you are compiling and using on the same computer then you should not need it. regards Garib On 4 Mar 2013, at 09:56, Adam Ralph wrote: Dear Ed, The error does indeed happen in ccp4lib. One of the first routines called by CCP4 progs is ccp4fyp. This initialises the CCP4 environment. See lib/cctbx/cctbx_sources/ccp4io/lib/src/ccp4_general.c. If you look at the code you can see that $CINCL is determined at run-time. You are right that this environment var is not needed at compile time. Files like environ.def and default.def are read at this time. Perhaps there has been a corruption of one of these files or you are pointing to an earlier version of $CINCL. Does the error occur with refmac alone or with every CCP4 prog? Adam Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac5 vs phenix refine mixed up
Dear Tim In principle if a user defines freer flag then refmac knows about that (unless freer flag is 0 then refmac assumes that it is default). In this case (if freer defined by user) then it is not altered. regards Garib On 25 Jan 2013, at 09:14, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Pavel, dear Garib, how do you figure out automatically the correct flag? (I hope both phenix and refmac will allow to manual overwrite the software's decision) Cheers, Tim On 01/24/2013 07:47 PM, Pavel Afonine wrote: Hi, It would be nice if default setting was the same in different suites. it's a nice idea of course, but I feel it is impractical as it would require changing a lot of software, both modern and legacy. However, given array of flags it is algorithmically trivial to figure out what is test and work flags. That's what phenix.refine have been doing since its beginning (2005). And my understanding is that Refmac does this too. As always, there are corner cases here, but it's better than nothing. Plus, programs (at least phenix.refine, can't speak for others) tell which flag was actually used, and they provide option to define the flag value to use. Pavel - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRAkzvUxlJ7aRr7hoRAlVfAKClRD4/JLNDcOab1HjBroQYXND3bQCfegA9 UiHvuKXg2/b3LqlbPWQpKmY= =Awum -END PGP SIGNATURE- Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac5 vs phenix refine mixed up
Dear all As it was already stated it is essential to use the same input file (after scaling and trancating) for all refinement sessions. Output mtz file in the absence of twinning has been scaled to account for anisotropic overall B values. It is modification of the data. In the twinning case output contains detwinned data. It is serious modification of the data and should not be used as input file for next refinement session. Output file in general is representation of the model and useful for model building but not for further refinement cycles. Regards Garib On 24 Jan 2013, at 11:30, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear, of course you could ask Garib whether or not the output data were modified by refmac5 - often they are, at least linearly scaled (which would certainly do no harm), and unless you have read the refmac5 code or Garib assures you I would not rely on it. Further trouble is that by using the output mtz-file, which contains more data columns like the sigma-weighted coefficients for map calculations, the e.g. GUI might accidentally pick the wrong one overlooked by the user, especially if the user is less experienced. To always use the same input mtz-file you avoid such possibilities and it also points a novice user to what refinement is actually doing. Best, Tim On 01/24/2013 12:03 PM, Qixu Cai wrote: Dear Tim Gruene, 2013/1/24 Tim Gruene t...@shelx.uni-ac.gwdg.de Dear Rajesh, first of all, a model is not true or false, it can only be better or worse. The explanation of what you observe depends on what you did: - did you use the identical and very same mtz-file as input to all three scenarios? Some people take the output mtz and use it as input to the next refinement cycle, which is a very, very, bad thing to do. Is the F/SIGF columns of the output mtz of refmac5 still the same as the F/SIGF columns of the input mtz? If they are the same, why cann't I use the F/SIGF columns of the output mtz as input to the next refinement? Thanks for your reply. - did you ensure always the same set of reflections was used for Rfree when switching between programs? If not, your R/Rfree are meaningless. It may also be that combining phenix and refmac5 indeed resulted in a better mode - both programs have some substantial differences in how they work. Best, Tim On 01/24/2013 11:12 AM, rajesh harijan wrote: Dear All, I am working on a perfectly twinned data in space group P31. when I refine this data with phenix refine the R/Rfree is 26.6/29.4 and average B-factor is 38. I did one test now. I used phenix refined pdb and refine with refmac5 and got R/Rfree of 26.2/29.7 and average B-factor is 64. Now I used refmac5 refined pdb and refined with phenix again. Now R/Rfree is 22.1/24.8 and average B-factor is 56. My question is, why B-factor gone up now and R/Rfree reduced. In which refined model should I believe in. If last refined model is true then how should I reduce the B-factor? Thank you Rajesh - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRARtHUxlJ7aRr7hoRAnvXAKCqUV5IHvKJShQHrN8/cCGmC4DDrACgw9gL 6MGqgIDK4DJ2vcHtuzdWPBc= =Pl4P -END PGP SIGNATURE- Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac5 vs phenix refine mixed up
Yes, Nat is right. Starting with the latest version 5.7 (that is part of ccp4) refmac makes sure that it uses correct set for free reflections. Hopefully it will remove some of the confusions when switching from one software to another. refmac 5.8 version should definitely have this feature. This version with some bug fixes and feature additions can be found from this page; http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/ This version should be available from the next ccp4 update. Garib On 24 Jan 2013, at 18:36, Nat Echols wrote: On Thu, Jan 24, 2013 at 10:34 AM, Leonid Sazanov saza...@mrc-mbu.cam.ac.uk wrote: Most likely scenario is that Phenix by default assigns Rfree flag as 1, while ccp4/refmac - as 0. That would explain your Rfree going down - because your Rfree reflections were refined by refmac. According to Garib, the current version of Refmac will automatically switch to the proper flags, so this problem should go away. -Nat Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Planar restraints in Refmac
Yes, it can be done. I may be able to do it soon. I have to find a better delimiter, + may not be good becuase it may be part of atom names. Another way would be: if you have more than 10-15 atoms then you can divide planes into several overlapping planes And yet another way (perhaps better way for large planes): describe planes with all possible local torsion angles with 0 or 180 degree target. regards Garib On 29 Nov 2012, at 10:36, andrea.p...@unina.it wrote: Thank you very much to all of you for your replies. The external restraints mechanism works perfectly as far as you do not exceed 468 characters in a string. If one wants to restraint more than let's say 15 atoms it is not feasible! Is it possible to list the atoms in a more compact way? I have in mind something like: first atom CA chain A residue 25 next atom CB+C+O+X1+X2 chain B residue 50 or something similar. Thank you again for your help. Andrea Quoting Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Andrea There are two ways as they were mentioned by Tim and Ian: 1) Using external restraints mechanism. You define something like (it is an example): external plane first atom CA chain A residue 25 next atom CB chain B residue 50 next atom OG chain B residue 100 next atom CC chain C residue 2 sigma 0.02 You need to add one line per plane. It is better to define these restraints in a file and then read in external keywords part of the refmac5 interface of ccp4i 2) Define link and use planes there. Links are defined for pairs of residues. The best way of defining links is using JLigand that can be downloaded from (it should be available from ccp4 6.3 also): http://www.ysbl.york.ac.uk/mxstat/JLigand/index.html there are very good tutorials written by Andrey Lebedev for new ligands as well as how to define links and use them in refmac regards Garib On 28 Nov 2012, at 07:03, Andrea Pica wrote: Hi everybody! Is there a simple way in REFMAC to restraints a group of atoms belonging to different residues to lay on a plane? Of course I would like to set the sigma! Do I have to add any line in the PDB header? Is it that simple? Thank you very much! Andrea Andrea Pica, Ph.D. student University of Naples Federico II Department of Chemical Sciences - Room 1N-04 Complesso Universitario Monte S. Angelo Via Cintia I-80126 Naples - Italy Phone 39-081 674269 Fax 39-081 674090 Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk Andrea Pica, Ph.D. student University of Naples Federico II Department of Chemical Sciences - Room 1N-04 Complesso Universitario Monte S. Angelo Via Cintia I-80126 Naples - Italy Phone 39-081 674269 Fax 39-081 674090 Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Planar restraints in Refmac
Dear Andrea There are two ways as they were mentioned by Tim and Ian: 1) Using external restraints mechanism. You define something like (it is an example): external plane first atom CA chain A residue 25 next atom CB chain B residue 50 next atom OG chain B residue 100 next atom CC chain C residue 2 sigma 0.02 You need to add one line per plane. It is better to define these restraints in a file and then read in external keywords part of the refmac5 interface of ccp4i 2) Define link and use planes there. Links are defined for pairs of residues. The best way of defining links is using JLigand that can be downloaded from (it should be available from ccp4 6.3 also): http://www.ysbl.york.ac.uk/mxstat/JLigand/index.html there are very good tutorials written by Andrey Lebedev for new ligands as well as how to define links and use them in refmac regards Garib On 28 Nov 2012, at 07:03, Andrea Pica wrote: Hi everybody! Is there a simple way in REFMAC to restraints a group of atoms belonging to different residues to lay on a plane? Of course I would like to set the sigma! Do I have to add any line in the PDB header? Is it that simple? Thank you very much! Andrea Andrea Pica, Ph.D. student University of Naples Federico II Department of Chemical Sciences - Room 1N-04 Complesso Universitario Monte S. Angelo Via Cintia I-80126 Naples - Italy Phone 39-081 674269 Fax 39-081 674090 Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac5 problem
Dear Cai On 5 Nov 2012, at 09:46, Qixu Cai wrote: Dear all, What's the difference between no prior phase information, phase and FOM, Hendrickson-Lattman coefficients, and SAD data directly in the refmac5 GUI of CCP4i? I would use SAD data directly if you have SAD data set. It just means that the program uses observed F+ and F- directly in refinement. This part of the program has been developed by Leiden group so you can direct your questions to them: Navraj S. Pannu r...@chem.leidenuniv.nl Pavol Skubak p.sku...@chem.leidenuniv.nl I am sure they will help you to get started. I have a SAD dataset and solve the phase by phenix.autosol. Now I want to refine the structure by refmac5, which kind of input above I should choose? Another question is in the Refinement Parameters of refmac5. What's the meaning of Use experimental sigmas to weight Xray terms? If I use molecular replacement to solve the structure, shall I uncheck this item? No. It is related with sigmas of observations and it is desirable to use them. Although there are some question marks how sigmas are estimated, they seemed to be better than nothing. regards Garib Thanks very much for your help. Best wishes, Q. Cai Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
[ccp4bb]
Dear all Could we stop at this point. regards Garib On 30 Oct 2012, at 18:35, Kavyashree Manjunath wrote: Dear Sir, I agree to that. But I presume that this is a platform to discuss scientific problems and not a forum to discuss or pour out personal frustrations. There may be other channels for such grievances but not this. I was just hoping this does not become a social network wherein everyone are free to express anything. Thank you kavya -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Kavya, given the test is written in proper English with proper grammer etc., I think you are actually asking for censorship. I am happy ccp4bb does not censor such emails, be they in accordance with one's opinion or not! Best, Tim On 10/30/2012 06:15 AM, Kavyashree Manjunath wrote: Dear CCP4 users, It is extremely sad that CCP4BB has failed to moderate/screen for such spam mails! Thanks kavya Dear Friends, There is no need to apply to this position, we suggest. It is a PREDETERMINED SELECTION, i.e. candidate is fixed and this (advertisement, screening, selection board, selection and approval) is just the procedure. It does not matter whether you apply or not. If you apply and called for interview, then you have to waste your valuable time as well as huge travel money unless some Big Boss is fixing you to the post. Interestingly Indian Institute of Science recruits and carries faculties and trains them in such a way that it has become a epicentre of recruitment scams across India and it make rest of Indian Scientists/Faculties in their path of scams and CRIME. Students also inherit the character of their boss. They do not participate in any form of fair selection in the country. Almost all cases they select and load many times inferior candidates even though candidate was not seen by anybody or interviewed. Similarly they distribute various national awards among themselves and within their group. THEY ARE NOT ASHAMED AT ALL. This is just an attempt of WASTING HUGE PUBLIC MONEY by a bunch of crooks who are good for nothing but worst for everything. Sham - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQj5K5UxlJ7aRr7hoRAum6AKDzlXQSoX827+OrPJOiWy1zF24pVgCgymMq Hgv5aAxCqVjSnONml1GSfx0= =KVPK -END PGP SIGNATURE- -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac
Hi Refmac use one letter code for RNA and two letter for DNA. Otherwise each residue is considered as one chain (if there is no link between them). I hope it helps. regards Garib On 31 Oct 2012, at 08:05, jp d wrote: hi, answering my own question, but maybe this will save some searching in the future, the error was related to 3 letter RNA codes refmac doesn't like that. jpd --- On Tue, 10/30/12, jp d yo...@yahoo.com wrote: From: jp d yo...@yahoo.com Subject: [ccp4bb] refmac To: CCP4BB@JISCMAIL.AC.UK Date: Tuesday, October 30, 2012, 3:43 PM hi, i have a large pdb file and i keep getting this error with refmac ERROR: number of chains 1500 i suspect something needs to be done to my pdb any suggestions ? thanks jpd Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] linking PLP-Lys
Dear Rajesh tutorial on this website is designed exactly for PLP-LYS links http://www.ysbl.york.ac.uk/mxstat/JLigand/index.html regards Garib On 23 Jul 2012, at 18:30, Rajesh Kumar wrote: Dear All, My friend needs a help. What is the best way to connect Lys to PLP with covalent bond. I am sure there are many ways do it. My friend would appreciate if you could simplify and explain this so that he could learn it without difficulties. Also I could learn I appreciate your time and help Thanks Rajesh Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Regarding refinement in Refmac5
1) Check free R flag. It may be related with free flag being 1 instead of 0. If it is the case then you can use newer version of refmac: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/ or from ccp4 v 6.3 2) Try arp/warp Garib On 19 Jul 2012, at 18:29, Deepthi wrote: Hi Yes i did check my MTZ file header. It shows p3221. For the same reason i reprocessed the data again in both p3221, p3121 and p321. Except for p3221 none of the other space groups fit the model. The packing looks good and the map shows side chains very clearly. When i add the respective side chain its just not accepting. May be it is not the correct solution? Is it possible? Thank You Deepthi On Thu, Jul 19, 2012 at 7:04 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: It isn't that your space group is wrong, but are you sure that your mtz file has that space group in its header? MR will test all possible alternatives - in this case P3121 or P3221 - but won't change the symmetry information in the input mtz. You need to do that with a utility like mtzutils hklin1 p3121.mtz hklout p3221.mtz SYMM P3221 end And there are various options in the REFLECTION UTILITIES. The integration and data processing are exactly the same for either space group, but REFMAC does not check that your mtz and pdb have the SAME symmetry information, and by default uses that in the mtz file. So you could have a perfectly good solution in P3221 but be running refinement in P3121, which is NOT GOOD! Eleanor. On 18 Jul 2012, at 17:50, Deepthi wrote: I tried opening the model with other spacegroups MTZ file. The map doesn't fit well for other spacegroups. The initial model was refined using Phenix Autobuild software. I tried MR with every spacegroup possible in primitive hexagonal. Only p3221 worked. There is no twinning in the crystal. I will try using other softwares for refinement but this is annoying. I also tried mutating the model to poly alanines and refine but this made it worse. The R-free went up to 0.546. I initially thought it might be a space group problem but trying other space groups doesn't work either. Thank youvery much for the help Deepthi On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi there, Not much information provided. How was the initial model refined ? Phenix ? It could be a problem with the Refmac refinement protocol (difficult to say with so little information) if you switched from Phenix to Refmac. How certain are you 1 - of the space group; 2 - that the crystal wasn't twinned ? You can have both and it can be annoying. Further, at this resolution I think you could use one of the SHELXes (forgot the terminology) for refinement, that could be more appropriate. F.V. Deepthi wrote: Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi -- Deepthi -- Deepthi Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Regarding refinement in Refmac5
Can you check space group in your mtz and pdb? I have seen this happening when they disagree. It is annoying and I would like it to be sorted out. If you want you can send your data and I can try to sort it out. Garib On 18 Jul 2012, at 17:50, Deepthi wrote: I tried opening the model with other spacegroups MTZ file. The map doesn't fit well for other spacegroups. The initial model was refined using Phenix Autobuild software. I tried MR with every spacegroup possible in primitive hexagonal. Only p3221 worked. There is no twinning in the crystal. I will try using other softwares for refinement but this is annoying. I also tried mutating the model to poly alanines and refine but this made it worse. The R-free went up to 0.546. I initially thought it might be a space group problem but trying other space groups doesn't work either. Thank youvery much for the help Deepthi On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi there, Not much information provided. How was the initial model refined ? Phenix ? It could be a problem with the Refmac refinement protocol (difficult to say with so little information) if you switched from Phenix to Refmac. How certain are you 1 - of the space group; 2 - that the crystal wasn't twinned ? You can have both and it can be annoying. Further, at this resolution I think you could use one of the SHELXes (forgot the terminology) for refinement, that could be more appropriate. F.V. Deepthi wrote: Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi -- Deepthi Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Rfactors stuck very high
270.0 51.590.0 180.0 90.0 -1. 0. 0. 2 2 90.0 90.0 90.0 51.545.0 90.0 180.0 0. 0.7072 0.7070 2 3 270.0 90.0 90.0 51.590.0 360.0 90.0 1. 0. 0. 2 4 270.0 90.0 270.0 51.545.0 270.0 180.0 0. -0.7070 0.7072 3 1 90.0 90.0 90.0 51.5 135.0 270.0 180.0 0. -0.7070 -0.7072 3 2 90.0 90.0 270.0 51.590.0 180.0 90.0 -1. 0. 0. 3 3 270.0 90.0 270.0 51.5 135.0 90.0 180.0 0. 0.7072 -0.7070 3 4 270.0 90.0 90.0 51.590.0 360.0 90.0 1. 0. 0. 4 1 270.0 90.0 90.0 51.590.0 360.0 90.0 1. 0. 0. 4 2 270.0 90.0 270.0 51.5 135.0 90.0 180.0 0. 0.7070 -0.7072 4 3 90.0 90.0 270.0 51.590.0 180.0 90.0 -1. 0. 0. 4 4 90.0 90.0 90.0 51.545.0 90.0 180.0 0. 0.7072 0.7070 Any suggestions and advice are very welcome. Cheers James Dr James Garnett Centre for Structural Biology Division of Molecular Biosciences Level 5 Sir Ernst Chain Building South Kensington Campus Imperial College London London SW7 2AZ Tel: +44 (0) 207 594 5464 Fax: +44 (0) 207 594 3057 Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] help regarding structure solution
Dear Sonali I would do the following things 1) Check your space group. Although rare it could be p2 or even p1 2) Run balbes server and check all space groups (in your case only p21 and p2) If you want you can send data to me to see what might be going on Regards Garib On 20 Jun 2012, at 19:13, sonali dhindwal wrote: Dear All, I am working on a protein for last so many years and for which i have got crystal now in a tray which i kept 1 years ago. It diffracts well and resolution is 2.2A, which is good. I indexed in HKL2000, mosflm and automar and it shows P21 space group in all data reduction packages. But when I tried using molrep or phaser then I do not get any solution. The sequence of my protein is having 46% identity with other available crystal structure. Also when I tried to get matthews coffecient, it calculates its molecular mass less ( about 35 kDa) than which should be (original 54kDa) with solvent content 47%. I have also run the silver staining gel of the protein which contained crystal that shows about 45 kD protein band which is 10 less than the original. Also I tried to run gel on crystal but it did not give anything as it was a small crystal. I have tried all combinations of the search model and tried to break available pdb many ways to make different search models but have not got any good solution. Molrep gives contrast even 10 or more but no good electron density map yet. Free R and figure of merit becomes 52% and 42% respectively in Refmac with all the solutions. I will highly appreciate all the suggestions for this kind of problem. Thanks and regards -- Sonali Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] correlations of B-factors and resolution
Please not that average B value may be arbitrary depending on data processing program, reference image etc. Average B values could be arbitrary (you can add to all atom single B value and it will not change information content of the data, they are removable). I think either variance of B or B - Bmin may correlate with resolution better. regards Garib On 16 May 2012, at 15:06, Nat Echols wrote: On Wed, May 16, 2012 at 6:46 AM, Qiang Chen c...@red.dfci.harvard.edu wrote: I have a 2.4A structure(pdb code 3LAF)with an average protein b-factor of 48. I wonder whether it's acceptable. Is there a direct correlation of b-factor and resolution? They're correlated, but it's not an exact relationship. See attached plot (which includes all entries in the PDB for which structure factors were deposited, as of about a year ago). The mean B-factor for structures between 2.3A and 2.5A resolution is just under 40, so your structure is very reasonable (the distribution is quite wide). The R and Rfree are 21.1% and 23.1%, respectively. This structure has a very high solvent content, 75%. Does it affect the b-factors? Only to the extent that high solvent content tends to result in more poorly ordered crystals and worse resolution - also, perhaps surface side chains will be more flexible if they're not forming crystal contacts. But it doesn't inherently change anything in refinement, aside from making it easier because you have a better observations-to-parameters ration. -Nat B_versus_d_min.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] How to refine CNS output file in refmac
Dear Vidisha The easiest way is to convert DNA names to standard pdb v3 names. You can do it using molprobity. The probelem is that GUA may have different meaning than G (RNA) or DG (DNA). regards Garib On 15 May 2012, at 16:37, tipu Lall wrote: Dear CCP4 users, I am a beginner in this field. It would be kind of all of you if can help me how to move ahead with my problem. I have an output file from CNS of a binary complex (DNA-protein) and I am trying to refine it with refmac5 (restrain refinement) and job is failing with error messages (written below) Before restrain refinement, I converted the CNS output file (pdb) from Codconv of CCP4 to pdb format. Thanks in advance Vidisha INFO: link is found (not be used) dist= 3.003 ideal_dist= 2.400 ch:CC res: 1 GUA at:C2' .-ch:Ca res: 2 GUA at:O2P . INFO: link is found (not be used) dist= 2.625 ideal_dist= 2.400 ch:CC res: 1 GUA at:C3' .-ch:Ca res: 2 GUA at:P . INFO: link is found (not be used) dist= 2.797 ideal_dist= 2.400 ch:CC res: 1 GUA at:C3' .-ch:Ca res: 2 GUA at:O2P . INFO: link is found (not be used) dist= 1.606 ideal_dist= 2.400 ch:CC res: 1 GUA at:O3' .-ch:Ca res: 2 GUA at:P . INFO: link is found (not be used) dist= 2.499 ideal_dist= 2.400 ch:CC res: 1 GUA at:O3' .-ch:Ca res: 2 GUA at:O1P . INFO: link is found (not be used) dist= 2.504 ideal_dist= 2.400 ch:CC res: 1 GUA at:O3' .-ch:Ca res: 2 GUA at:O2P . INFO: link is found (not be used) dist= 2.507 ideal_dist= 2.400 ch:CC res: 1 GUA at:O3' .-ch:Ca res: 2 GUA at:O5' . INFO: link is found (not be used) dist= 2.843 ideal_dist= 2.400 ch:CC res: 1 GUA at:O3' .-ch:Ca res: 2 GUA at:C5' . INFO: link is found (not be used) dist= 2.637 ideal_dist= 2.400 ch:Ca res: 2 GUA at:C3' .-ch:Cb res: 3 ADE at:P . INFO: link is found (not be used) dist= 3.116 ideal_dist= 2.400 ch:Ca res: 2 GUA at:C3' .-ch:Cb res: 3 ADE at:O1P . INFO: link is found (not be used) dist= 1.604 ideal_dist= 2.400 ch:Ca res: 2 GUA at:O3' .-ch:Cb res: 3 ADE at:P . INFO: link is found (not be used) dist= 2.504 ideal_dist= 2.400 ch:Ca res: 2 GUA at:O3' .-ch:Cb res: 3 ADE at:O1P . INFO: link is found (not be used) dist= 2.514 ideal_dist= 2.400 ch:Ca res: 2 GUA at:O3' .-ch:Cb res: 3 ADE at:O2P . ATTENTION: atom:O4 GUA 1 CC is missing in the structure ATTENTION: atom:O3 GUA 1 CC is missing in the structure ATTENTION: atom:C3 GUA 1 CC is missing in the structure ATTENTION: atom:C1 GUA 1 CC is missing in the structure ATTENTION: atom:O1 GUA 1 CC is missing in the structure ATTENTION: atom:O2 GUA 1 CC is missing in the structure === Error: Fatal error. Cannot continue Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] pdb and cif file generation from smiles string
Is that what you are looking for?libcheck cn generate it (JLigand should be able). grade should also generate from smiles.Garib 1.cif Description: Binary data 1.pdb Description: Binary data On 9 May 2012, at 17:08, Shya Biswas wrote:Hi all,I am having trouble generating a pdb and cif file from the following smiles string:O=C(C[N+]23CN1CN(CN(C1)C2)C3)c45c45Prodrg fails to run when i draw the molecule in JME editor was wondering if anyone knows a better program which does this kind of job. thanks in advance,shya Dr Garib N MurshudovGroup Leader, MRC Laboratory of Molecular BiologyHills RoadCambridgeCB2 0QH UKEmail:garib@mrc-lmb.cam.ac.ukWebhttp://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] pdb and cif file generation from smiles string
Interesting: N+ does not seem to be chiral. Three out of four carbons attached to it seem to be equivalent. Garib On 9 May 2012, at 18:13, Shya Biswas wrote: The following seems to work with phenix: O=C(C[N@+]23CN1CN(CN(C1)C2)C3)c45c45 Shya On Wed, May 9, 2012 at 12:15 PM, Bosch, Juergen jubo...@jhsph.edu wrote: Is that your molecule ? Screen Shot 2012-05-09 at 12.13.18 PM.png On May 9, 2012, at 12:08 PM, Shya Biswas wrote: O=C(C[N+]23CN1CN(CN(C1)C2)C3)c45c45 .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Powder Rings in Single Crystals
As far as I know there are several bumps: around 3.5-4 (there are some at low resolution related with molecular shapes also) - secondary structures, ~2.2 related with angles and around 1.2 related with covalent bonds. For DNA/RNA there is one more bump around 1.6-1.7 ( I thought that is because of Phosphor bonds). They are visible with normalised data better. I think there was a paper by Morris and Bricogne in Acta Cryst about these things. These bumps have some consequences - B values calculated using Wilson plot usually jump up and dow around these bumps. Garib On 9 May 2012, at 19:44, Nat Echols wrote: On Wed, May 9, 2012 at 11:35 AM, Edward A. Berry ber...@upstate.edu wrote: Still I would expect to see peaks in a wilson plot around bond-length resolution, similar to the peaks due to secondary structure at lower resolution. I was curious about this myself, so I looked at the Wilson plot for an atomic-resolution structure (attached). There is at best a small hump around 1.5A; I suspect uncertainties in atomic positions (i.e. the B-factor) reduce the effect. This was consistent in several other atom-resolution datasets (of different proteins). I'm curious what the bump around 2.25A is. -Nat wilson_highres.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Powder Rings in Single Crystals
Yes, in principle, on paper it is possible. Moreover in many cases by looking at the various directional Wilson plots you may be able to see direction of helices (just like in DNA/RNA). However in general case it is a little bit tricky (mixture of different secondary structures directed in different directions, noisy data etc). And there is another complication that bonds are involved in intensity curves via sinc function that is not completely local. I think there were attempts (by Hamburg group) with some success. What happened to their publication, I do not know. You are right that in molecular replacement they are implicit. However if bonds are directed more or less in the same direction then assumption behind Wilson distribution breaks down and it may affect molecular replacement (not as severe as pseudo translation, nevertheless serious enough). Fortunately most molecular replacement programs are forgiving for such departures from assumptions. regards Garib On 9 May 2012, at 20:28, Jacob Keller wrote: It seems to me that spherical forms of Wilson plots could be used to determine how many bonds of what nature were oriented in which direction, and this may have been what Bricogne's micro molecular replacement technique was capitalizing on? For example, one might be able to orient a straight DNA molecule by finding the direction at which the ~3.2-3.5 Ang bin signal was greatest. But I guess this is probably implicit in molecular replacement anyway... JPK On Wed, May 9, 2012 at 2:08 PM, Nat Echols nathaniel.ech...@gmail.com wrote: On Wed, May 9, 2012 at 11:58 AM, Garib N Murshudov ga...@mrc-lmb.cam.ac.uk wrote: As far as I know there are several bumps: around 3.5-4 (there are some at low resolution related with molecular shapes also) - secondary structures, ~2.2 related with angles and around 1.2 related with covalent bonds. For DNA/RNA there is one more bump around 1.6-1.7 ( I thought that is because of Phosphor bonds). They are visible with normalised data better. It has been pointed out to me that my example cut off the data at too low a resolution to see the peak for covalent bonds. Here is a different version that shows a distinctive peak around 1.15A. -Nat -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refining beta peptides
If they are short then you can create links between amino acids and use them. Please have a look tutorials for jligand: http://www.ysbl.york.ac.uk/mxstat/JLigand/ You need only one link and declare it between peptides. I think buster, phenix and refmac use same type of dictionaries. All they may be able to handle these links. I think in shelxl you need to define all bonds, there may be a program to help to design them, but I am not qualified to give any recommendation on that. If you have long beta-peptides then adding a lot of links might be tedious. Then we need to modify our programs (in our case refmac) to handle these links automatically. If you have any question or need help to defining links and using them within ccp4 then please let me know. regards Garib On 21 Apr 2012, at 23:34, Richard Baxter wrote: Dear All, Looking to refine a beta-peptide structure (2 A resolution). Any advice on use of Refmac, Phenix, SHELX for this, are they in the dicionary? Thanks a lot. Richard -- Asst. Prof. in Chemistry = Yale University PO Box 208107 New Haven, CT 06520-8107 Tel: +1 203 432 9576 Fax: +1 203 432 6144 www.baxterlab.org = Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Yes there is. If you use command line (it is not available on the ccp4i yet). If you run with command lines refmac5 all others like hklin, xyzin etc mskout mask file name eof all options you want eof Then there will be a map and you can visualise it using coot. regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] bulk solvent treatment inside protein cavities
A follow up: In the new version there is FC_ALL_LS, PHIC_ALL_LS That should be FC_ALL_LS = FC + FMASK. I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC But it is after scaling. If you write out mask map then it is just 0 1 map (0 inside protein and 1 outside), except values are not 0 1 but 0 and some constant Regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] bulk solvent treatment inside protein cavities
Dear Ketaro At the moment mskout option is a signal that the program should stop. Obviously I can add an option to continue. However if you have mskout option it is likely that you want to check what is going on with the mask. If you want to compare starting and final mask then you could run refmac with mskout in the beginning and after refinement. If you need it urgently then I can add continuation of refinement with mskout option. FC_ALL is ML scaled FC+FMASK Sometime it may be different from least-squares scaled FC+FMASK regards Garib On 16 Apr 2012, at 16:01, Keitaro Yamashita wrote: Dear Garib, Thank you very much for your quick reply. I tried mskout option and the output looked almost the same as the map generated by FC_ALL - FC. By the way, when mskout option is specified, refmac stops before CGMAT cycles. Is there any way to do refinement with mskout option? I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC What is FC_ALL in the new version? Thanks, Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: A follow up: In the new version there is FC_ALL_LS, PHIC_ALL_LS That should be FC_ALL_LS = FC + FMASK. I have not tried but if you can use vector difference map then it should be: FMASK = FC_ALL_LS - FC But it is after scaling. If you write out mask map then it is just 0 1 map (0 inside protein and 1 outside), except values are not 0 1 but 0 and some constant Regards Garib On 16 Apr 2012, at 15:09, Keitaro Yamashita wrote: Dear Garib, Is there REFMAC option to output solvent mask information (e.g. Fmask and PHImask in mtz to check with Coot)? I tried to generate it by subtracting (FC, PHIC) from (FC_ALL,PHIC_ALL). But I'm not sure that FC_ALL = FC + FMASK is correct or not. Keitaro 2012/4/16 Garib N Murshudov ga...@mrc-lmb.cam.ac.uk: Dear Allister Could you please update refmac version. In the version you it seems that bulk solvent mask calculation has some problems. New version (at the moment) can be downloaded from this site: http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.7_linux.tar.gz There is a mac version also. regards Garib On 16 Apr 2012, at 11:37, Allister Crow wrote: Board members, I have a couple of questions regarding how to improve the solvent model as applied to solvent-filled cavities inside proteins. I am currently nearing the end of refinement of a protein structure at 2.8 A resolution. I recently switched Refmac versions, upon doing this I noticed a modest improvement in R factors, but I also notice some new features in the difference maps. These features don't show up in the sigma-weighted 2Fo-Fc maps and are unlikely to be 'ligands' of any form. In fact, I suspect that the appearance of these features (which are all located in solvent channels within cavities inside the protein) are probably due to some difference in how the bulk solvent contribution has been applied. I've attached a picture of one such feature showing the difference between Refmac 5.5 and 5.6. (Both difference maps are contoured at 3 sigma- both using the same model and refinement parameters). My questions are therefore: 1) has something substantial changed in the bulk solvent treatment between Refmac versions 5.5 and 5.6? 2) How can I go about changing the bulk solvent treatment to better account for solvent contribution inside the protein cavities? Best wishes, and thanks in advance for all your help, - Allister Crow bulk_solvent_inside_cavities.png Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
Dear Chris Could you please try later version of refmac then if the problem persists please let me know. Before making any suggestions it would be good to make sure that the problem is not related with particular software version (as Ian suggested) regards Garib On 4 Apr 2012, at 16:16, Chris Meier wrote: Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] P21, twinned date NCS refinement
I would say that you should use ncs restraints in any case. NCS relates atoms and twin relates intensities. In some sense presence of twinning reduces information contents (in the limiting case the number of (effective) observervations becomes twice less) of the data and using NCS decreases the effective number of adjustable parameters. Without NCS some of the stats (like RvR) cannot be trusted. However you should remember that if ncs is very close to twin operators then in reciprocal space they relate two reflections exactly and in most of the cases you do not gain much and differences between ncs related molecules might be more important than their similarity. I would suggest using local ncs then global differences are retained and molecules are made locally similar. If bet angle is 92.4 degrees (I would also check obliquity in refmac output if you are using that. In your case this number should be non-zero, meaning that twin related reflections will not coincide exactly) then I would expect splitting of spots in some directions at high resolutions. One should be careful when integrating, if only one of these spots are integrated then you may end up have several classes of reflections: low resolution and in some directions when spots overlap perfectly you have contributions from two crystals, in split spot cases each spot has contribution from single crystal. regards Garib On 27 Mar 2012, at 21:37, Bosch, Juergen wrote: Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both anyhow) to the experts out there here's my question: We have a P21 dataset with 2 molecules in the asu and a refined twin fraction of 38% according to phenix.refine using a twin law operator. My gut feeling tells me that I can't use NCS unless I detwin the data, is that a correct assumption ? How does phenix.refine [PDB] [mtz] twin_law=h,-k,-l main_ncs=true would deal with this problem ? Same question goes to Garib, it's very convenient to just specify twin in your Refmac script without further values and magic happens but what if I add NCS restrains, will Refmac treat them correctly according to the twin operator ? Twins are confusing in real life and even more in crystals I think. And no the data can not be processed in a higher space group, P222 results in Rmerges of 40% in the lowest resolution shell, the beta angle is 92.4 degrees in P21. And if processed in P1 we get 4 molecules per asu and a refined twin fraction of 50%, which in my eyes clearly indicates it's not P1 but really P21. Hope to get some interesting feedback on this issue. Thanks, Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] P21, twinned date NCS refinement
Unless you have very good reason it is better to use highest possible space group (without going over). Then you do not have problem of related reflections and covariances between them, If you go to P1 then reflection will be related with crystallographic as well as NCS as well as twin symmetries. When you go to p21 then you at least do not have to deal with reflections related with crystallographic symmetry. I am not sure going to P1 would solve problem of split reflections. It is a fundamental problem in data integration programs that assume there is only one lattice with one orientations. Your example shows that reality is a little bit more complicated. Split spots will cause problems in profile generations and in theory will affect quality of the data also. If you could you should try to inegrate split spots as one (merge them) then the problem becomes similar to merohedral twinning (when spots overlap exactly). Regards Garib On 27 Mar 2012, at 22:00, Bosch, Juergen wrote: Thanks Garib for your input. And yes we do see some split spots. We used XDS to overcome (I hope) most problems but still intensities of perfectly overlapping reflections will be too large. Would you think it's safer to integrate the data in P1 as symmetry mates will not be merged and then solve in P1 and convert into P21 cell for further refinement afterwards ? Jürgen On Mar 27, 2012, at 4:53 PM, Garib N Murshudov wrote: I would say that you should use ncs restraints in any case. NCS relates atoms and twin relates intensities. In some sense presence of twinning reduces information contents (in the limiting case the number of (effective) observervations becomes twice less) of the data and using NCS decreases the effective number of adjustable parameters. Without NCS some of the stats (like RvR) cannot be trusted. However you should remember that if ncs is very close to twin operators then in reciprocal space they relate two reflections exactly and in most of the cases you do not gain much and differences between ncs related molecules might be more important than their similarity. I would suggest using local ncs then global differences are retained and molecules are made locally similar. If bet angle is 92.4 degrees (I would also check obliquity in refmac output if you are using that. In your case this number should be non-zero, meaning that twin related reflections will not coincide exactly) then I would expect splitting of spots in some directions at high resolutions. One should be careful when integrating, if only one of these spots are integrated then you may end up have several classes of reflections: low resolution and in some directions when spots overlap perfectly you have contributions from two crystals, in split spot cases each spot has contribution from single crystal. regards Garib On 27 Mar 2012, at 21:37, Bosch, Juergen wrote: Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both anyhow) to the experts out there here's my question: We have a P21 dataset with 2 molecules in the asu and a refined twin fraction of 38% according to phenix.refine using a twin law operator. My gut feeling tells me that I can't use NCS unless I detwin the data, is that a correct assumption ? How does phenix.refine [PDB] [mtz] twin_law=h,-k,-l main_ncs=true would deal with this problem ? Same question goes to Garib, it's very convenient to just specify twin in your Refmac script without further values and magic happens but what if I add NCS restrains, will Refmac treat them correctly according to the twin operator ? Twins are confusing in real life and even more in crystals I think. And no the data can not be processed in a higher space group, P222 results in Rmerges of 40% in the lowest resolution shell, the beta angle is 92.4 degrees in P21. And if processed in P1 we get 4 molecules per asu and a refined twin fraction of 50%, which in my eyes clearly indicates it's not P1 but really P21. Hope to get some interesting feedback on this issue. Thanks, Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1
Re: [ccp4bb] REFMAC5 residues with bad geometry
Hi David Occupancis of input file are very suspicious and not all atoms of resides are present, some occupancies are zero. In refmac zero occupancy means it does not exist. It may explain the problem In refinement we could add an option to make occuancies one if there are no alts but it would be dangerous thing to do. Since occupancies are suspect I would check input pdb file carefully. regards Garib On 24 Mar 2012, at 18:39, David Schuller wrote: CCP4 6.2.0 Refmac_5.6.0117 Scientific Linux 6.1 In my current model, I notice that several sidechains are falling apart, despite having gone through a few rounds of refinement with REFMAC5 and model building with COOT. The worst examples were all Glu and Arg residues. I tried switch to the REFMAC5 executable on the updates page, which was Refmac_5.6.0114, with no obvious difference. Eventually I noticed that these are all residues containing atoms with occupancy less than 1.00, which must be a carry over from the MR search model. I set all the occupancies to 1.00 and this seems to have fixed the problem. This seems counter-intuitive to me. If the occupancies are set low, shouldn't the geometry restraints be stronger relative to the density refinement? Cheers, ATOM 1479 N GLU A 7 -51.844 -33.605 37.318 1.00 60.26 N ATOM 1480 CA GLU A 7 -53.137 -33.849 37.966 1.00 59.28 C ATOM 1481 CB GLU A 7 -52.997 -33.664 39.476 1.00 61.37 C ATOM 1482 CG GLU A 7 -52.799 -32.212 39.905 0.48 60.42 C ATOM 1483 CD GLU A 7 -53.349 -32.573 41.635 0.00 54.47 C ATOM 1484 OE1 GLU A 7 -52.557 -31.998 42.106 0.83 52.26 O ATOM 1485 OE2 GLU A 7 -55.014 -32.911 42.408 0.68 50.75 O ATOM 1486 C GLU A 7 -54.293 -32.985 37.412 1.00 62.61 C ATOM 1487 O GLU A 7 -55.444 -33.240 37.737 1.00 63.42 O ATOM 3165 N ARG A 77 -46.032 -33.003 26.272 1.00 55.82 N ATOM 3166 CA ARG A 77 -44.959 -32.368 27.071 1.00 60.92 C ATOM 3167 CB ARG A 77 -44.050 -31.428 26.231 1.00 54.56 C ATOM 3168 CG ARG A 77 -42.702 -31.102 26.892 1.00 69.21 C ATOM 3169 CD ARG A 77 -42.278 -29.628 26.867 0.46 63.93 C ATOM 3170 NE ARG A 77 -41.587 -29.303 25.625 0.79 61.76 N ATOM 3171 CZ ARG A 77 -41.607 -28.610 24.146 0.00 37.32 C ATOM 3172 NH1 ARG A 77 -43.177 -26.956 23.467 0.85 60.52 N ATOM 3173 NH2 ARG A 77 -41.267 -28.245 23.427 0.95 58.82 N ATOM 3174 C ARG A 77 -45.585 -31.698 28.281 1.00 64.89 C ATOM 3175 O ARG A 77 -45.949 -32.377 29.262 1.00 77.93 O -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Refinement with pseudo-translation
Dear Shiva I would be careful with omit maps in the presence of pseudotranslation. You have to omit bits of molecule from PST related molecules simultaneously, otherwise you will have bias. If you look at the equation it becomes clear why. R factors 50/53 is close to random (in the presence of solvent random R factors seem to be around 53). If you want you can send your data and we can try to have a look. I cannot promise anything but at least you can have a second opinion regards Garib On 22 Mar 2012, at 22:28, Shiva Kumar wrote: Dear CCP4bb members I have a 3.0 Å dataset which has an off-origin peak of height 36% in patterson map. The peak is at fractional co-ordinates 0, 0.5, 0.5. Data has been indexed in P2(1)22(1) SG using HKL2000. I have located all the molecules in asu (as far as I know) using Molrep with the 'locked rotation' and 'Use' PST feature. After 1 round (20 cycles) of rigid body and 1 round (10 cycles) of restrained refinement (Refmac), the R and Rfree are 49 and 53 %. Although the R factors are very high, I feel the solution might be correct because the electron density follows c-alpha trace in almost all places. To be sure, I deleted a beta strand from the structure's core and repeated the refinement and found that the electron density for the strand was still present (I have no experimental phases). I have the following questions: 1) Are the final R factors never expected to reduce to acceptable values given the 36% off-origin peak? 2) What is the best way to settle the SG? I was considering P2(1)2(1)2(1), P2(1)22, P2(1)2(1)2 and P2(1)22(1), considering the off-origin peak is at 0,0.5,0.5. I found all the molecules in asu only using the P2(1)22(1) data in Molrep. Is this the best way to settle my SG? 3) Some CCP4bb archives advise either refining against weak and strong reflections alternatively (https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1010L=ccp4bbD=01=ccp4bb9=AJ=ond=No+Match%3BMatch%3BMatchesz=4P=204490) or refining against medium intensity reflections. Should I also be doing these things? If yes, then what is the best way of doing it? Your suggestions and corrections to my interpretation of our data would be appreciated. Regards Shiva Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refining phosphorylated residues
In new version (it should be in ccp4 6.2.0, if not then it will come ccp4 6.3, otherwise you can take it from York's web site: ) TPO as well as SEP are peptides. Break in coot may be due to misinterpretation of SEP or TPO as peptide in coot and it may be because of older version of the dictionary. Do the distances, angles between neighbouring residues make sence? If not then refinement also had problems regards Garib On 22 Mar 2012, at 00:39, Joel Tyndall wrote: As a follow up question the bulletin board, why is SEP a peptide (L-peptide) and TPO not (non-polymer)? Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rajesh kumar Sent: Thursday, 22 March 2012 12:00 p.m. To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] refining phosphorylated residues Dear All, I have a structure of a protein and peptide complex, in which peptide has modified residues ( phosphoserine and phosphothreonine). During refinement these both gets disconnected with adjacent residues and its hard to connect them. Could you please suggest me some options. Thanks Rajesh Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Refmac version
Dear Kavya In principle you should be able to use newer version for old pdb file unless you have pdb v2 namings for DNA/RNA etc. New version of ccp4 has more dicitionary elements (1 or so). Older version was compiled for 3000. That is the reason why old version does not work with new dictionary. regards Garib On 19 Mar 2012, at 10:10, Kavyashree M wrote: Dear users, I was using Refmac 5.5.0102 (ccp4- 6.1.2) for refining the structures, I was supposed to do one more round of refinement with final model but unfortunately system crashed and i had to install the new version of ccp4 (6.2.0) which has refmac-5.6.0117. So my doubt here is - can I do the refinement of the final model with this version of refmac or do I need to complete with the older version itself? I however tried replacing the older version of refmac in place of new refmac. But the job failed while running (in the new interface) with an error message ERROR: number of monomers 3000 /lib. limit/ Change parameter MAXMLIST in lib_com.fh Do i need to use the old interface itself or can i use the new interface to run old refmac binary? Kindly provide some suggestion. Thanking you With Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] MolRep error
you can try a version from this site: http://www.ysbl.york.ac.uk/~alexei/molrep.html If it does not work please let me know regards Garib On 15 Mar 2012, at 12:45, Justyna Wojdyla jw...@york.ac.uk wrote: Dear All, I am trying to run MolRep with 'input fixed model' option and i am getting error message: At line 1260 of file /usr/local/xtal/ccp4-6.2.0/src/molrep_/molrep.f Fortran runtime error: End of record I am using: CCP4 6.2: MOLREP(ccp4) version 11.0.02 : 07/02/11 I found the updated version of the molrep binary for Windows system but could not see anything for linux. I'll be grateful for your help. Justyna -- Dr Justyna Aleksandra Wojdyla Department of Biology University of York Heslington, York, YO10 5DD United Kingdom Tel: +44 (0)1904 328818 Email: justyna.wojd...@york.ac.uk EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] MR and pseudo-translationnal-symmetry
There are 3 options: 1) Use molrep. If you have two copies related with PST then it can give right solution 2) Take the latest version of phaser and use it. It can deal with PST with two related copies also 3) Reduce cell dimensions (it does not seem to be possible in your case. PST is not exactly on cell edges. But you can try). Work in c2 with smaller cell and then expand. but before going into all these trouble make sure that you really have PST. Calculating patterson (ccp4i, fft) and displaying using coot can give you some insight. Relation between origin and non-origin peaks can give you some insight. regards Garib On 13 Mar 2012, at 10:21, vincent Chaptal wrote: Dear ccp4, I have a case of PTS and wonder what's the best strategy to handle my data. I processed my data in C2 with a=161 b=109 c=225 beta=104. The data in 97% complete to 3,8A. xtriage detected a 40% peak in the patterson at fractional coordinates x=-0,001 y=0,055 z=0,5. I want to try to phase using MR. Should I: - leave the data in C2 (fully complete) and specify the program to use PST. - expand from C2 to P1 and run using PST. - re-index in P1 (a=98 b=97 c=225, alpha=78 beta=78, gamma=68) with only ca. 80% completeness, and specify PST. before shooting more crystals to increase compleness and going for HA phasing, i was wondering if i could do something with what i have. thank you for your input. vincent -- Vincent Chaptal, PhD Institut de Biologie et Chimie des Protéines Drug-resistance modulation and mechanism Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01 http://www.ibcp.fr Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Help! weird thing
Hi Could you please check: 1) If there is psedotranslation. It could be done by using sfcheck, molrep, ctruncate or calculating patterson map and displaying using coot at 8-10 sigma level (it is my favourite method for analysis of pseudo translations), whole unit cell ( a bit bigger than whole unit cell). Then if you see large no origin peak (very likely along one of the axis, could be a). If yes then you have several options: using phaser - 1) reduce cell, find solution in smaller cell and then expand; 2) use molrep to solve. When there are two copies related with pseudo translation molrep can give you solution; 3) as far as I am aware latest version of phaser works with pseudo translation. If you have pseudtranslation you should be aware that even if you solve the structure starting R factors could be 70-80%. Then you may want to do 40 cycles of rigid body and 40-100 cycles of ljelly body 2) Check your space group in pdb and mtz file. They may not be consistent. I hope it helps. Garib On 11 Mar 2012, at 07:33, xiaoyazi2008 wrote: Hi All, I have an interesting thing to share. 2.3A dataset with good quality, P21 Partial model is available (~60% of the target protein). It seems that there are 4 copies in the ASU (Matthews_coef 2.6, 53%solvent) Molecular replacement gave two copies of the model (Z scores are R6.2, T6.2, R6.8, T13.4). The solution is very clear. It could not locate the rest two copies. However, a quick refmac5 refinement gave a very high R factor. The funny part is the symmetry operation in Coot. As shown in the JPEG figure, it looks like there should be another two copies (based on strong fo-fc green map), which locate in the empty space between models found by Phaser. Why is that Phaser could not find the remaining two copies even there are strong fo-fc density? Any suggestions... Thanks a lot! Zhihong weird thing.jpg Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] REFMAC Riding Hydrogens
Hi 1) refmac's default option has been generate all starting from version 5 (it is around 14 years). The reason is for most tests generating hydrogens gave good R or geometry. And if you look at the original paper by Ramachandran (I think it was pulished in 1963) you see using hard ball approximation and including all atoms generated plot is very similar to what we know as Ramachandran plot. A little bit more work on included hydrogen plus phi/psi torsion angle restraints (in free form, not from Ramachandran plot) should generate faithfully Ramchandran plot. It needs to be tested properly and carefully. refmac version 4 did not have hydrogen generation option. 2) ccp4i's option has always been use hydrogens if present in file (I personally do not like this option: this option should be used with care) 3) If there is no non-bondedn ... others then it is very likely that hydrogens were not used. 4) I am not sure that there is a fullproof way to know if hydrogens were added or not. Regards Garib On 7 Mar 2012, at 15:00, Paul Smith wrote: Hello CCP4bb, Firstly, thanks to all for your comments. However, I'm still unsure how to sort all of this riding hydrogen business out. Robbie's comments seem particularly apt: Because there were some reporting errors in the past (http://proteincrystallography.org/ccp4bb/message18808.html) it is hard to tell from the PDB when refinement with hydrogens became hip. Is there any foolproof way to know if a recently deposited file was refined with riding hydrogens in REFMAC, especially since some such structures do not yet have publications associated with them? How about the value of NON-BONDED CONTACTS OTHERS? If that is other than NULL, does that mean riding hydrogens were present? Also, how about refmac version numbers? Is there a clear demarcation when riding hydrogens a) became available, b) became default, or c) became default in the CCP4i GUI? Thanks again, --Paul From: Robbie Joosten robbie_joos...@hotmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, March 6, 2012 4:26 AM Subject: Re: [ccp4bb] REFMAC Riding Hydrogens Hi Everyone, Pavel’s statement is likely a bit of an exaggeration, but he has a valid (yet hard to prove point). The default in CCP4i was (and is?) to use hydrogens only if present in the input file. This is IMO not a safe default. Because there were some reporting errors in the past (http://proteincrystallography.org/ccp4bb/message18808.html) it is hard to tell from the PDB when refinement with hydrogens became hip. Discussions on this BB show that at the use of riding hydrogens is still not fully accepted, especially at low resolution (where they actually help most). Cheers, Robbie From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel Afonine Sent: Monday, March 05, 2012 21:53 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] REFMAC Riding Hydrogens Dear Tim, good catch, thanks; I could craft that phrase more carefully! Although often it may not be quite fair to take phrases out of context: this newsletter article was written in the context of macromolecular refinement. And yes, recently may be a broad term -:) All the best, Pavel On Mon, Mar 5, 2012 at 12:45 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Pavel, you may want to add to the structures mentioned in [1] one or two organic structures present in the Cambridge Database. Until recently it was customary to ignore hydrogen atoms throughout the process of crystallographic X-‐ray structure determination. [1] 'recently' as in 1997 [2]? Even though 1997 is probably a poor estimation of the corresponding year... Cheers, Tim [1] On contribution of hydrogen atoms to X-ray scattering http://www.phenix-online.org/newsletter/ [2] http://shelx.uni-ac.gwdg.de/SHELX/shelx.pdf On 03/05/2012 09:14 PM, Pavel Afonine wrote: Hi, On Mon, Mar 5, 2012 at 11:52 AM, Matthew Franklin mfrank...@nysbc.orgwrote: Adding the riding hydrogens generally gives you some improvement in R factors even with a good quality (i.e. stereochemically correct) model. and here are the results of more or less systematic test that prove this: see On contribution of hydrogen atoms to X-ray scattering here: http://www.phenix-online.org/newsletter/ Pavel - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPVSXkUxlJ7aRr7hoRAm1TAJ9Hyfhkl3yhD5QSKw9I4RSK58m0fACgmlxk YGILzeMam/3gQVmCeh0vQ8k= =3m2J -END PGP SIGNATURE- Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http
Re: [ccp4bb] REFMAC Riding Hydrogens
On the interface under Refinement Parameters there are three options 1) Use if present 2) Do not use 3) Generate all if you are using script then default is generate all (add them riding positions). You can switch to other options: make hydr all# generate all - default option make hydr yes # use if present in the file make hydr no # do not use Controlling output make hout yes # write hydrogens to the output file make hout no # do not write - default optoon I hope it helps regards Garib On 5 Mar 2012, at 19:15, Paul Smith wrote: Hello CCP4 community, I'm posting at large regarding a previously raised issue for REFMAC for which I cannot find the conclusion in the old threads. Specifically, does REFMAC add riding hydrogens during default refinement? Though I've not requested that any hydrogens be added, only used if in the input, I see the following in the output header: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS Does anyone know exactly what this remark is referring to? Are riding hydrogens added for stereochemical restraints, or are their scattering contributions calculated regardless of they are explicitly defined? Can the hydrogen behavior in REFMAC be more explicitly controlled. Thanks, --Paul Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] twin refinement in refmac
1) You should use measured data (after scala/aimless/truncate). In general there may not be one to one relationship between observed data and asymmetric unit (e.g. non-merohedral twinning) and it would not be possible to bring input data to output file. Use original data 2) Internally refmac groups reflection into classes. All twin related reflections belong to one class. In the example you give four reflections belong to one class. FreeR is property of the class not individual relfections. I.e. all twin related reflections belong either to free or working class. If your input data has this property then output should also have this. In the output file there will be 0 (for free) and 1 (working) 3) At early stages it is not easy to factorise twin and underestimated symmetry. It seems that L-test is the best way of making decision if you crystals are twinnined. If you collect data from many crystals and merge them then you artificially twin (or increase twinning). Using pointless to index all datasets consistently may sort some of the problems 4) In this case it seems that you may need to reindex. Largest twin domain is not the first one Regards Garib On 2 Mar 2012, at 02:00, wtempel wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Generating parameters/cif files for macrocyclic ligands
Hi Joel Herman is right: If you are refining cyclic peptides then the easiest way is to use link record linking C-terminus with N terminus. the name of the link should be TRANS. Here is an example: LINK ALA S 21 ASN S 1TRANS It will force ALA 21 to be linked (with torsion, angles, planes, bonds etc) to ASN 1 of chain S. This way you do not have to create description for large molecule. If you still want to create one molecule and you have mol2 file with coordinates then you can use libcheck to generate full dictionary using following commands libcheck file_mol mol_file_name nodist y It should generate fdescription. However I would prefer using link record. this way you keep amino acid names etc intact. If you amino acids are not among existing then you will need to create their description first and declare them peptide. regards Garib On 8 Feb 2012, at 10:33, herman.schreu...@sanofi.com wrote: Hi Joel, The way I solved this problem was by generating a linear peptide and then connecting the ends using a LINK card in the header of the pdb. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Joel Tyndall Sent: Tuesday, February 07, 2012 10:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Generating parameters/cif files for macrocyclic ligands Hi folks, I have an intriguing problem. I’m trying to generate a cif file for a macrocyclic peptide (of the likes in pdb1d4k). They are cyclic tripeptides units. I can generate a pdb or mol2 file easily. I have used PRODRG to generate a .cif file and Coot read thjis in nicely. However, as it is cyclic one cannot adjust the dihedral angles. I have previously done this using CNS where you can break the tricyclic peptide into residues and generate parameters to specify bonds/links between the residues (which allows this kind of movement). I can’t come up with a way to do this without using CNS. I have looked ta J-ligand which allows for one link “between” two separate residues which precludes a macrocycle. I have looked at sketcher within CCP4 which reads the pdb files but I don’t believe this can be done here. Within Coot I can refine the whole ligand but not certain components. Any suggestions greatly appreciated . ( I may stick to coot refinement with fixed atoms at this stage) Regards Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] New restraints, same name
That is the plan and hopefully with help of people from pdb coot, buster, phenix, refmac, ccp4 programs will all use mmcif with ligand descriptions inside. Good will is there, a little bit work is needed. Regards Garib On 25 Jan 2012, at 08:15, Ingo P. Korndoerfer wrote: ... and why don't we move on from pdb format to something a bit more modern ? i think it would only require a handful of programmers to agree on this and the rest would (have to) follow. i.e. if coot and refmac could both read and write something more modern we'd have a huge part of ccp4 world covered. are we thinking of ourselves at working at the frontiers of science or not ... (... o.k. ... different topic, but you get the idea ...) ingo On 25.01.2012 03:53, Paul Emsley wrote: Thanks to all contributors, I have been informed, educated and entertained. A bit of background perhaps... (it seems that I have been living in the 0.7 world long enough to forget that not everyone else is here). [T]he viewer programs don't care about the restraint dictionaries says Seth Harris - haha - in olden Coots that was the case... :) It is my hope that Coot will be used for comparison, evaluation, validation and manipulation of ligands in protein-ligand complexes and their electron density. My current obsession is with chemical structure diagrams - here's a recent screenshot: http://lmb.bioch.ox.ac.uk/coot/screenshots/Screenshot-example-2010-01-02.png ... and here's one I made earlier today, illustrating the sorts of problems I am trying to handle (PI3 Kinase ligand, 4a55): http://lmb.bioch.ox.ac.uk/coot/screenshots/Screenshot-Coot-prodrg-valence-problem.png amusing, eh? Anyway, to make the chemical diagram and the 3D bonding representation I need to construct a description of the ligand that contains bond orders. Hence restraints. So yes, let me emphasize that this is mostly for drawing pictures and I don't see the use case of refinement of multiple different ligand complexes as very useful. I do like Dale's idea - the use of HETNAM and synonyms - so, as I understand it, the PDB file has a residue called LIG and the dictionary has a comp-id of 2-(N-methylmethanesulfonamido)-6-(propan-2-yl)pyrimidine (or XYZ0123456 or whatever) and a HETNAM record in the PDB file provides the mapping. Is this a solution? It is attractive because it requires very little work from me. I did consider Judit's idea, i.e. check the atom names in the coordinates against the dictionary before bonding. I thought that there may be (too many?) pathological cases where the names did match (at least for ligand fragments) but the chemistry did not. Let me know if you think that I need not worry so much about that. This is relatively easy to do. However, this only solves the tangle problem - and I think that that for practical purposes that may be covered now as I have recently turned off restraints auto-loading for several special three-letter codes - one can (at least) see noddy bonding instead of a tangle. To answer Garib's point: yes, in Coot there is indeed a single table/dictionary of restraints, with the key/index being the comp-id/residue-name. It applies to all molecules. I had not before considered the option of embedding monomer restraints inside a Coot molecule - that might be a cleaner solution. I will ponder on that. It does mean that you will have to read restraints after reading coordinates though. And yes, I do occasionally wonder how computational chemistry software (Maestro, Vida for example?) solves this problem. Presumably such software is used to show several overlaying ligand structures (all called LIG?). And computational chemists like to see chemistry, and not just coloured sticks, right? Thanks, Paul. Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] New Faster-than-fast Fourier transform
After quick look at the manuscript: It is applicable to sparse signals (i.e. number of non-zero elements is not whole space). It would be applicable to inverse FFT after density modification and gain would not be that much. k-sparse approximation would loose signal (strictly speaking it does not produce exact FFT but an approximation, i.e. very weak signals (electron density, fourier coefficients) may be ignored) At the moment I am a bit sceptical about its use in crystallography. Although with careful implementation twice speed up crystallographic FFT could be possible. Garib On 20 Jan 2012, at 17:37, Jim Fairman wrote: New Fourier transform algorithm supposedly improves the speed of Fourier transforms get up to a tenfold increase in speed depending upon circumstances. Hopefully this will get incorporated into our refinement programs. http://web.mit.edu/newsoffice/2012/faster-fourier-transforms-0118.html -- Jim Fairman, Ph D. Crystal Core Leader I/Project Leader I Emerald BioStructures Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] R/Rfree problem
You can try zanuda from York's site. www.ysbl.york.ac.uk/YSBLPrograms/index.jsp It may help. It may be that you have a twinned crystal. As far as I know best available test for twinning is L-test (truncate produces this test). When you go from P1 to P212121 if L-test shows increased twinning then it may mean that you are overmerging. regards and I hope it helps Garib On 19 Jan 2012, at 12:06, 조기준 wrote: Dear all, I have a data, and it has R/Rfree problem. The data could be processed as P212121 and P1. The resolution was 2.9, and unit cell parameters were a=73.527, b=90.035, c=237.980, α=β=μ=90 and a=73.709, b=90.099, c=238.172, α=89.939, β=89.945, μ=89.993 for P212121 and P1, respectively. R/Rfree was 22.3/29.4 for P1 after several refinements. However, I couldn’t decrease R/Rfree below 30.0/37.6 for P212121. There were almost no differences on monomer structure and crystal packing between P212121 and P1 after MR, and the electron densities followed trace of peptide chain well. Can anybody suggest me about this problem? Thank you. Ki Joon Cho Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Reference for JLigand
Hopefully by the time your paper accepted it will be out. Here is the reference: JLigand: a graphical tool for CCP4 template restraint library Lebedev AA, Young P, Isupov MN, Moroz OV, Vagin AA and Murshudov GN (2012) Acta Cryst D68, in press (submitted, in consideration or whatever) I hope it helps regards Garib On 18 Jan 2012, at 09:47, Pedro M. Matias wrote: Dear CCP4 Developers, What is the correct reference for JLigand to include in a publication ? Thanks best regards, Pedro Matias Industry and Medicine Applied Crystallography Macromolecular Crystallography Unit ___ Phones : (351-21) 446-9100 Ext. 1669 (351-21) 446-9669 (direct) Fax : (351-21) 441-1277 or 443-3644 email : mat...@itqb.unl.pt http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit Mailing address : Instituto de Tecnologia Quimica e Biologica Apartado 127 2781-901 OEIRAS Portugal Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] How to tell Refmac it is a fixed double bond?
Dear Sam double bond is only used to find bon lengths and other parameters. Refmac uses bond length. As I see you would like to make a link between two residues. There is a tutorial written by Andrey Lebedev that addresses exactly this type of problems. Please have a look this site: http://www.ysbl.york.ac.uk/mxstat/JLigand/index.html there are three tutorials: 1) simple ligand, 2) links (that is what you want) and 3) simple metal containing ligand treatment. I hope it helps. If it does not please let us know. regards Garib On 18 Jan 2012, at 21:07, Sam Arnosti wrote: Hi every one I have made a Cif file for the restraints of my ligand with Jligand, which is attached to my protein via a lysine-aldehyde Schiff base formation. The problem is that whenever I run the refmac with the Cif file with torsions and link description, it changes the distance of the Lysine and the Carbon of my ligand. The density is there, but it does not recognize it as a C=N bond and puts them up to 2 angstrom away from each other. I do not know, if I should change the link description to make the Refmac regonize the double bond or what else I can do. This is the description of link in my Cif file: _chem_link_bond.link_id _chem_link_bond.atom_1_comp_id _chem_link_bond.atom_id_1 _chem_link_bond.atom_2_comp_id _chem_link_bond.atom_id_2 _chem_link_bond.type _chem_link_bond.value_dist _chem_link_bond.value_dist_esd LYS-MER 1 NZ 2 C18 double 1.2600.020 I appriciate your help beforehand. Regards Sam Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] JLigand distorts molecules
That is interesting. Do you use the latest ccp4 and dictionary that comes with it? What are the version of dictionary, libcheck, refmac Just typing refmac5 -i libcheck -i you should get version information. The version of the dictionary is on the top of a file head $CLIBD_MON/list/mon_lib_list.cif ccp4 version should be 6.2, refmac either 5.6 or after 5.7.007 libcheck 5.2 Dictionary 5.28 or later. regards Garib On 13 Jan 2012, at 10:13, Wolfgang Skala wrote: Dear Garib, thanks for your reply. However, when I use the new versions, 3GP is an apparently random polyhedron which does not resemble 3'-GMP at all. Yours Wolfgang Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] JLigand distorts molecules
That is interesting. It works for me here and for few other people in other places. Can you exit and restart JLigand, can you send me a figure of what is happening? regards Garib On 13 Jan 2012, at 11:00, Wolfgang Skala wrote: ccp4 is 6.2.0, refmac5 is 5.7.0010 (the file you provided; formerly 5.6.0117), libcheck is 5.2 (formerly 5.1.14), dictionary is 5.28 I also tried each of the four refmac5-libcheck combinations, but without success. yours Wolfgang Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] JLigand distorts molecules
In the current version there is a bug and we have fixed it (as far as i know). It is not JLigand bug, it is libcheck bug. If you take the version (5.7 version) then it may work better. http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/ You need refmac5.7_linux.tar.gz or refmac5.7_macintelt.tar.gz After gunzipping you need to copy refmacgfortran and libcheckgfortran to $CBIN. It would be better if you keep older version so that you can change back if something happens cp $CBIN/refmac5 $CBIN/refmac5.6 cp refmacgfortran $CBIN/refmac5 cp $CBIN/libcheck $CBIN/libcheck5.6 cp libcheckgfortran $CBIN/libcheck If it also does not work please let me know Cheers Garib On 12 Jan 2012, at 12:43, Boaz Shaanan wrote: Hi, You are right. I happen to have 1.0.7 lying around and there 3GP is fine. A bug must have been introduced somewhere on the way between versions. I wonder whether it's only 3GP. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Wolfgang Skala [wolfgang.sk...@sbg.ac.at] Sent: Thursday, January 12, 2012 1:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] JLigand distorts molecules Dear colleagues, has anyone of you experienced problems regarding regularization in JLigand? When I start the first tutorial and load 3'-GMP (3GP), the double bond to the O6 atom is about twice as long as the remainder of the molecule; the rings are also highly distorted. Similarly, if I modify a ligand, regularization yields a structure that is definitely out of shape. I'm using JLigand 1.0.25 on Ubuntu 11.10 and tried both the current version of OpenJDK and Sun Java JRE 6u30; even compiling the JLigand source on my platform with Sun Java SE 6u30 did not help. Kind regards, Wolfgang Skala -- Structural Biology Group / Department of Molecular Biology University of Salzburg Billrothstraße 11 5020 Salzburg Austria Phone: +43 662 8044 7278 http://www.uni-salzburg.at/xray Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac Bsol
Dear Berhnard It seems there are only one or two questions remain to clarify. On 31 Dec 2011, at 07:20, Bernhard Rupp (Hofkristallrat a.D.) wrote: Dear Garib, thank you for the quick response despite (or because of) the holidays. I’ll try to summarize because I am not sure I understand yet, and it might be useful for all : Ø Partial structure mask bulk solvent parameters. Mask bulk solvent B value is in addition to protein B value. Not quite clear what you are saying here – how does that answer what the ‘Overall’ means versus the ‘Partial’? (some guessing below) Overall in the pdb header is calculated from atominc B values. Ø Full scaling is like this: Fscaled = scale_protein exp(-B s^2/4) exp(-s^T U s) (F_protein + scale_mask exp(-B_mask s^2/4) Fmask) (1-scale_babinet exp(-B_babinet s^2/4)) Ok then I see that I can use either _mask or _babinet terms because only one set of terms is in effect if the other one is zero. If I turn both off, I get basic k, B scaling, plus anisotropic B correction. Makes perfect sense. Based on that I will try to determine which line in the printout is actually the bulk solvent contribution. For the ‘Partial structure’ part: Partial structure1: scale =0.375, B = 24.388 If I recall correctly, for the flat bulk solvent model the scale_mask and B_mask are generally in the order of 0.4 and 40AA, so then this ought to be scale_mask and B_mask of the solvent contribution? Yes? No? Now for Partial is mask bulk solvent. Overall : scale =0.891, B = -0.266 Now it gets interesting: what is this in terms of the above equation? I don’t seem to be able to factor out a single overall scale and B correction from your eqn, but it sure looks like a correction term from bulk to be applied to the overall k and B…. Can you clarify please? Again: Overall B value in the pdb header is from atominc B values. Bvalue here is remaining part. It should be 0 but because of differences between lsq scaling and ML it is not always 0. For refinenemnt we use ML and for scaling/Rfactor calculations ls scaling. Ø Current version of refmac does not calculate Wilson B value I think we can get that readily from say truncate. But that won’t be in the PDB REMARK 3 then… Yes. It would not be in the pdb header after refmac. It needs to be sorted out. I.e. refmac should calculate/read and write tot the pdb header Ø Note: Overall Bvalue may correspond to residual overall B value if you are using TLS refinement Understood. Ø If you use simple scaling then mask solvent is still on. Yes – that is consistent with the doc. Ø You can turn it off by Calculate the contribution of from the solvent region Yes, that can be done by unclicking the box Calculate the contribution from the solvent region. Handy if one needs to check certain things… Ø To turn Babient's bulk solvent you need to use Babinet's scaling instead of simple. Yep, understood. Thx, BR Regards and Happy New Year Garib From: Garib Murshudov [mailto:garib.murshu...@gmail.com] On Behalf Of Garib N Murshudov Sent: Thursday, December 29, 2011 4:47 PM To: b...@hofkristallamt.org Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refmac Bsol Partial structure mask bulk solvent parameters. If you use simple scaling then mask solvent is still on. You can turn it off by Calculate the contribution of from the solvent region To turn Babient's bulk solvent you need to use Babinet's scaling instead of simple. I hope it helps regards Garib Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac Bsol
Partial structure mask bulk solvent parameters. If you use simple scaling then mask solvent is still on. You can turn it off by Calculate the contribution of from the solvent region To turn Babient's bulk solvent you need to use Babinet's scaling instead of simple. I hope it helps regards Garib On 29 Dec 2011, at 21:08, Bernhard Rupp (Hofkristallrat a.D.) wrote: Dear Refmac developers group, I am trying to understand a small detail in the refmac log listing. In the bulk solvent section just below the restraint table (I use monitor MANY in general): - Overall : scale =0.891, B = -0.266 Partial structure1: scale =0.375, B = 24.388 Overall anisotropic scale factors B11 = -0.39 B22 = 0.09 B33 = 0.33 B12 = -0.00 B13 = 0.48 B23 = 0.00 - Is the listing indeed containing the flat bulk solvent model (‘simple model’ in refmac gui, SOLVENT YES) k and B in the first 2 lines? If so, I am curious a) what exactly is ‘Overall’ versus ‘partial structure’? What is the relative magnitude of the scale and B telling me in each case? b) would it not be useful to have it reported in the PDB header? All I find there is the anisotropic B scaling matrix and a mean (overall) B value (of unspecified origin – how exactly is that computed?) REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2) : NULL REMARK 3 MEAN B VALUE (OVERALL, A**2) : 18.711 Explanation/intuition would be much appreciated. Best regards, BR - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 hofkristall...@gmail.com b...@hofkristallamt.org http://www.hofkristallamt.org/ -- Old and treacherous will beat young and skilled every time -- PS: The doc leaves me a little confused because SCAL type SIMP would imply KB = K0*exp(-B0*s^2) (Simple Wilson scaling) i.e. K1 = 0 while BULK (Babinet) which I did not specify KB = K0*exp(-B0*s^2) * (1- K1*exp(-B1*s^2)) gives me 2 Ks and Bs. So I conclude that ‘overall’ and ‘partial’ lines do not list Babinet ks and Bs (the B is also too small for that) Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
Check your Rfree flag. Phenix and refmac may use different flag. Have a look into log file. If percentage of Rfree reflections is 95% then flags need to be swapped. Garib On 9 Dec 2011, at 02:50, Mark J van Raaij wrote: are you sure that you are using the original input intensities for the REFMAC map calculation (and the refinement run) and not the output ones of the (previous) run? if you are not, you may have model bias, and Rfree can be fooled, especially with NCS (if you have it). - or perhaps anisotropic B-factor refinement (if you are using it), works better in REFMAC than PHENIX? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 18:36, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] jumping ligand
Does it happens when you use automatic NCS restraints? On 30 Nov 2011, at 18:40, Ed Pozharski wrote: Did anyone ever seen a ligand molecule (or water, maybe) moved into a symmetry-related position upon refinement in refmac? If that is a feature (e.g. to make sure that non-protein stuff is coordinated to the spot of the closest contact), how can I disable it? Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] negative density in difference map
There may be several reasons. 1) Artefacts (some of them) a) effect of mask: if there are large holes inside molecule and there should be no electron density (for example hydrophobic holes) then in older versions masks would put a constant density there and as a result difference map would have negative densities. This has been dealt with in the newer version b) Effect of TLS refinement. If you are using TLS then you can try without TLS and compare densities. If it is effect of TLS then without TLS you should not see negative density. TLS model is a simple model assumes that all atoms in the group oscillates as a rigid group. Loops and other flexible parts may not obey this assumption 2) Genuine density Alternative conformations (Met almost always are in more than one conformations). In these cases you may be able to see alternative conformation if you look at very low level of electron density. And you may be able to see if it is case by looking at the B values. If neigbouring atoms have large differences in B values then it may be because of alt conformation Regards Garib On 23 Nov 2011, at 07:54, Careina Edgooms wrote: Good morning CCP4 members I have a question about a 2F0-Fc difference map that I calculated with Refmac. In some instances it gives me negative (red) density around part of a side chain and no positive density in sight. Furthermore the entire residue fits well into the blue density of the complete map, including the part with negative density. I am struggling to interpret this. Does the fact that it fits the blue density mean that the side chain is in the correct place or does the red blob on part of the side chain (eg on the sulphur in a Met residue) mean that something funky is happening with this side chain? Thanks for any assistance Careina Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] ions in REFMAC refinament
You do not have to change dictionary. You can specify charges on atoms using element columns in the pdb file. For example: ATOM139 O HOH A 35 13.139 -4.487 5.983 1.00 31.43 AA1 O ATOM140 CL CL A 35 13.139 -7.487 5.983 1.00 31.43 AA1 CL-1 Regards Garib On 1 Nov 2011, at 22:47, Ivan Shabalin wrote: Dear Refmac users, Im a bit confused how Refmac treats ions. In the monomers library I can find Cl.cif: CL . 'chlorine' non-polymer 1 1 . # data_comp_CL # loop_ _chem_comp_atom.comp_id _chem_comp_atom.atom_id _chem_comp_atom.type_symbol _chem_comp_atom.type_energy _chem_comp_atom.partial_charge CLCL CL CL0.000 It does not have charge and atom.atom_id is CL, but not Cl-1 (I believe, it is important for the scattering factor). I tried to change these fields but refmac crushed. Searching through CCP4BB I found that Ions from nonions are distinguished by just addition charge on the element column in the PDB file. So, for Cl I have to type Cl-1 in the last field of PDB-file line. I tried and it worked, at least REFMAC uses different coefficients for atomic scattering factor for the ion and B-factors are changed a bit. But I never seen in PDB files that somebody specifies Cl-1 or Mg+2. And when you bring new ligand with Coot id does not specify the charge. I have two questions: 1) Cl and Cl- have very different atomic radii and the scattering factor coefficients are quite different. But, does it really make significant difference for refining? 2) Is it possible to specify atom type in the library, so that the scattering factor coefficients will be taken into account by Refmac? Or changing PDB-file manually is the only way? Thank you very much for any comments!! With best regards, Ivan Shabalin, Ph.D. Research Associate, University of Virginia 4-224 Jordan Hall, 1340 Jefferson Park Ave. Charlottesville, VA 22908 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] RE : [ccp4bb] refmac 5.6 ccp4 6.2.0
Hi All I think that is the reason. In version of refmac (with new dictio uses new pdb v3 hydrogen naming and there are a lot of differences. regards Garib On 28 Oct 2011, at 10:00, LEGRAND Pierre wrote: Hi Kenneth, I am experiencing exactly the same problem, in similar conditions: refinement with H at 0.84 A resolution. From a bunch of tests made yesterday, I have found that it is linked to incompatibilities between cif dictionaries definitions and H names in the input PDB. For me, one simple solution to that problem, was to remove all H atoms from my input pdb and let refmac rebuild them (MAKE HYDR ALL). I hope the trick will work for you. By the way, I am facing an other problem: Clashes in inter residues H-bonds between H and acceptor atoms. What is the correct way to define these. The HYDBND pdb definition doesn't seems to work. Do I need to use LINK definitions ? Is there a way to do that automatically ? Cheers, Pierre De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Robbie Joosten [robbie_joos...@hotmail.com] Date d'envoi : vendredi 28 octobre 2011 09:42 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] refmac 5.6 ccp4 6.2.0 Hi Kenneth, This looks like an off-by-one bug in the restraint generation. Typical sources are weird LINKs, wrong atom names and bad luck. I suggest you make sure you have the very latest Refmac and dictionary and try setting up a new refinement instead of recycling an old job. If that doesn't work, we may need a closer look at your input model. Cheers, Robbie Date: Thu, 27 Oct 2011 20:48:49 -0500 From: satys...@wisc.edu Subject: [ccp4bb] refmac 5.6 ccp4 6.2.0 To: CCP4BB@JISCMAIL.AC.UK Has anyone had problems with Refmac 5.6? I tried refining our stucture at 1.24 A, aniso with H in riding position and it just exploded! I get error in distances such as Standard External All Bonds: 3270 0 3270 Angles: 4923 0 4923 Chirals: 214 0 214 Planes: 368 0 368 Torsions: 957 0 957 --- Number of reflections in file 90428 Number of reflections read 90428 CGMAT cycle number = 1 Bond distance outliers Bond distance deviations from the ideal 10.000Sigma will be monitored A 5 PRO C A - A 5 PRO HB1 A mod.= 3.344 id.= 1.329 dev= -2.015 sig.= 0.014 A 5 PRO C B - A 5 PRO HB1 B mod.= 2.997 id.= 1.329 dev= -1.668 sig.= 0.014 A 5 PRO HB1 A - A 5 PRO HG1 A mod.= 2.292 id.= 1.458 dev= -0.834 sig.= 0.021 A 5 PRO HB1 A - A 6 LEU HD23A mod.= 7.407 id.= 0.860 dev= -6.547 sig.= 0.020 A 5 PRO HB1 B - A 5 PRO HG1 B mod.= 2.247 id.= 1.458 dev= -0.789 sig.= 0.021 A 5 PRO HB1 B - A 6 LEU HD23B mod.= 6.529 id.= 0.860 dev= -5.669 sig.= 0.020 A 5 PRO HG1 A - A 5 PRO HD1 A mod.= 2.267 id.= 0.980 dev= -1.287 sig.= 0.020 A 5 PRO HG1 A - A 6 LEU N A mod.= 4.860 id.= 1.530 dev= -3.330 sig.= 0.020 A 5 PRO HG1 A - A 6 LEU HD21A mod.= 6.129 id.= 1.525 dev= -4.604 sig.= 0.021 A 5 PRO HG1 B - A 5 PRO HD1 B mod.= 2.236 id.= 0.980 dev= -1.256 sig.= 0.020 A 5 PRO HG1 B - A 6 LEU N B mod.= 4.922 id.= 1.530 dev= -3.392 sig.= 0.020 A 5 PRO HG1 B - A 6 LEU HD21B mod.= 6.664 id.= 1.525 dev= -5.139 sig.= 0.021 A 6 LEU N A - A 6 LEU CA A mod.= 1.467 id.= 0.970 dev= -0.497 sig.= 0.020 A 6 LEU N A - A 6 LEU HA A mod.= 2.005 id.= 0.970 dev= -1.035 sig.= 0.020 A 6 LEU N A - A 6 LEU CB A mod.= 2.497 id.= 1.530 dev= -0.967 sig.= 0.020 A 6 LEU N B - A 6 LEU CA B mod.= 1.469 id.= 0.970 dev= -0.499 sig.= 0.020 A 6 LEU N B - A 6 LEU HA B mod.= 2.032 id.= 0.970 dev= -1.062 sig.= 0.020 A 6 LEU N B - A 6 LEU CB B mod.= 2.446 id.= 1.530 dev= -0.916 sig.= 0.020 A 6 LEU CB A - A 6 LEU HB2 A mod.= 0.969 id.= 1.521 dev= 0.552 sig.= 0.020 A Rfree goes form 17 to 28 and R from 15 to 25. Coot map looks like a bunch of busted insect parts. I use the exact same input using ccp4 6.1.13 and Refmac 5.5 and all is good. I am forced to use the old ccp4 and refmac to publish. Rf 17 R 15. thanks -- Kenneth A. Satyshur, M.S.,Ph.D. Associate Scientist University of Wisconsin Madison, Wisconsin 53706 608-215-5207 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] RE : [ccp4bb] refmac 5.6 ccp4 6.2.0
SIGNATURE- Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] off topic: Adobe demos deblur of photos...
Position (general motion blur is a special case of it) dependent blurring can be applied to denisty improvement but problem is extremely ill posed. While deblurring you need to reduce noise amplification. Proper regularisation needs to be designed, probem becomes NxN linear equation where N is the number of grid points in density which might be a little bit large. However iterative method exists in inverse problems field. regards Garib On 11 Oct 2011, at 19:37, Pete Meyer wrote: I could be wrong, but my understanding is that they're removing motion blur from the image - so I don't think it'll be directly applicable to density modification. But I'd be very happy to be wrong on this one. Pete Francis E Reyes wrote: http://hoowstuffworks.blogspot.com/2011/10/adobe-demos-amazing-unblur-feature.html Though I can't really see the image myself... the gasp of the audience is telling With respect to existing density modification programs, I wonder if such technology (whatever it is) can ever clear up my messy density maps. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Ice rings... [maps and missing reflections]
In the limit yes. however limit is when we do not have solution, i.e. when model errors are very large. In the limit map coefficients will be 0 even for 2mFo-DFc maps. In refinement we have some model. At the moment we have choice between 0 and DFc. 0 is not the best estimate as Ed rightly points out. We replace (I am sorry for self promotion, nevertheless: Murshudov et al, 1997) absent reflection with DFc, but it introduces bias. Bias becomes stronger as the number of absent reflections become larger. We need better way of estimating unobserved reflections. In statistics there are few appraoches. None of them is full proof, all of them are computationally expensive. One of the techniques is called multiple imputation. It may give better refinement behaviour and less biased map. Another one is integration over all errors (too many parameters for numerical integration, and there is no closed form formula) of model as well as experimental data. This would give less biased map with more pronounced signal. Regards Garib On 11 Oct 2011, at 20:15, Randy Read wrote: If the model is really bad and sigmaA is estimated properly, then sigmaA will be close to zero so that D (sigmaA times a scale factor) will be close to zero. So in the limit of a completely useless model, the two methods of map calculation converge. Regards, Randy Read On 11 Oct 2011, at 19:47, Ed Pozharski wrote: On Tue, 2011-10-11 at 10:47 -0700, Pavel Afonine wrote: better, but not always. What about say 80% or so complete dataset? Filling in 20% of Fcalc (or DFcalc or bin-averaged Fobs or else - it doesn't matter, since the phase will dominate anyway) will highly bias the map towards the model. DFc, if properly calculated, is the maximum likelihood estimate of the observed amplitude. I'd say that 0 is by far the worst possible estimate, as Fobs are really never exactly zero. Not sure what the situation would be when it's better to use Fo=0, perhaps if the model is grossly incorrect? But in that case the completeness may be the least of my worries. Indeed, phases drive most of the model bias, not amplitudes. If model is good and phases are good then the DFc will be a much better estimate than zero. If model is bad and phases are bad then filling in missing reflections will not increase bias too much. But replacing them with zeros will introduce extra noise. In particular, the ice rings may mess things up and cause ripples. On a practical side, one can always compare the maps with and without missing reflections. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Ice rings... [maps and missing reflections]
Best way would be to generate from probability distributions derived after refinement, but it has a problem that you need to integrate over all errors. Another, simpler way would be generate using Wilson distribution multiple times and do refinement multiple times and average results. I have not done any tests but on paper it looks like a sensible procedure. regards Garib On 11 Oct 2011, at 20:58, Ethan Merritt wrote: On Tuesday, October 11, 2011 12:33:09 pm Garib N Murshudov wrote: In the limit yes. however limit is when we do not have solution, i.e. when model errors are very large. In the limit map coefficients will be 0 even for 2mFo-DFc maps. In refinement we have some model. At the moment we have choice between 0 and DFc. 0 is not the best estimate as Ed rightly points out. We replace (I am sorry for self promotion, nevertheless: Murshudov et al, 1997) absent reflection with DFc, but it introduces bias. Bias becomes stronger as the number of absent reflections become larger. We need better way of estimating unobserved reflections. In statistics there are few appraoches. None of them is full proof, all of them are computationally expensive. One of the techniques is called multiple imputation. I don't quite follow how one would generate multiple imputations in this case. Would this be equivalent to generating a map from (Nobs - N) refls, then filling in F_estimate for those N refls by back-transforming the map? Sort of like phase extension, except generating new Fs rather than new phases? Ethan It may give better refinement behaviour and less biased map. Another one is integration over all errors (too many parameters for numerical integration, and there is no closed form formula) of model as well as experimental data. This would give less biased map with more pronounced signal. Regards Garib On 11 Oct 2011, at 20:15, Randy Read wrote: If the model is really bad and sigmaA is estimated properly, then sigmaA will be close to zero so that D (sigmaA times a scale factor) will be close to zero. So in the limit of a completely useless model, the two methods of map calculation converge. Regards, Randy Read On 11 Oct 2011, at 19:47, Ed Pozharski wrote: On Tue, 2011-10-11 at 10:47 -0700, Pavel Afonine wrote: better, but not always. What about say 80% or so complete dataset? Filling in 20% of Fcalc (or DFcalc or bin-averaged Fobs or else - it doesn't matter, since the phase will dominate anyway) will highly bias the map towards the model. DFc, if properly calculated, is the maximum likelihood estimate of the observed amplitude. I'd say that 0 is by far the worst possible estimate, as Fobs are really never exactly zero. Not sure what the situation would be when it's better to use Fo=0, perhaps if the model is grossly incorrect? But in that case the completeness may be the least of my worries. Indeed, phases drive most of the model bias, not amplitudes. If model is good and phases are good then the DFc will be a much better estimate than zero. If model is bad and phases are bad then filling in missing reflections will not increase bias too much. But replacing them with zeros will introduce extra noise. In particular, the ice rings may mess things up and cause ripples. On a practical side, one can always compare the maps with and without missing reflections. -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] How to get formfactor for Zn +2.
If you will put element names in correct positions then refmac may have a chance to find it. Here is corrected positions: ATOM 1893 O HOH A 258 -8.934 52.268 49.467 0.00 66.53 O ATOM 1894 ZNZN B 1 -10.456 38.580 26.267 1.00 57.36 ZN+2 ATOM 1895 O HOH C 1 -5.932 42.917 25.589 1.00 24.02 O In your case all element names are moved towards left and B value of ZN is in incorrect position also. ATOM 1893 O UNK A 258 -8.934 52.268 49.467 0.00 66.53 O ATOM 1894 ZN+2 ZN B 1 -10.456 38.580 26.267 1.00 57.36ZN+2 ATOM 1895 O HOH C 1 -5.932 42.917 25.589 1.00 24.02 O Regards Garib On 4 Oct 2011, at 15:31, Eleanor Dodson wrote: Can anyone advise me how to get the Zn+2 formfactor from atomsf.lib The input coordinate is given atom type ZN+2 but the formfactor is that for Zn: I changed the atom name to Zn+2 but that made no difference... ATOM 1893 O UNK A 258 -8.934 52.268 49.467 0.00 66.53 O ATOM 1894 ZN+2 ZN B 1 -10.456 38.580 26.267 1.00 57.36ZN+2 ATOM 1895 O HOH C 1 -5.932 42.917 25.589 1.00 24.02 O refmac log says: loop_ _atom_type_symbol _atom_type_scat_Cromer_Mann_a1 _atom_type_scat_Cromer_Mann_b1 _atom_type_scat_Cromer_Mann_a2 _atom_type_scat_Cromer_Mann_b2 _atom_type_scat_Cromer_Mann_a3 _atom_type_scat_Cromer_Mann_b3 _atom_type_scat_Cromer_Mann_a4 _atom_type_scat_Cromer_Mann_b4 _atom_type_scat_Cromer_Mann_c N 12.2126 0.0057 3.1322 9.8933 2.0125 28.9975 1.1663 0.5826 -11.5290 C 2.3100 20.8439 1.0200 10.2075 1.5886 0.5687 0.8650 51.6512 0.2156 O 3.0485 13.2771 2.2868 5.7011 1.5463 0.3239 0.8670 32.9089 0.2508 SE17.0006 2.4098 5.8196 0.2726 3.9731 15.2372 4.3543 43.8163 2.8409 S 6.9053 1.4679 5.2034 22.2151 1.4379 0.2536 1.5863 56.1720 0.8669 ZN14.0743 3.2655 7.0318 0.2333 5.1625 10.3163 2.4100 58.7097 1.3041 Eleanor Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] How to get formfactor for Zn +2.
I think it is a pdb rule. Here are some of the rules: http://www.wwpdb.org/documentation/format33/sect9.html Charges (confusingly) could be 2+ etc. Regards Garib On 4 Oct 2011, at 16:41, Eleanor Dodson wrote: OK So the ATOM TYPE has ZN O C S etc in column 77/78 and the +2 etc in 79/80 Is there any documentation for this? E On 10/04/2011 03:56 PM, Garib N Murshudov wrote: If you will put element names in correct positions then refmac may have a chance to find it. Here is corrected positions: ATOM 1893 O HOH A 258 -8.934 52.268 49.467 0.00 66.53 O ATOM 1894 ZNZN B 1 -10.456 38.580 26.267 1.00 57.36 ZN+2 ATOM 1895 O HOH C 1 -5.932 42.917 25.589 1.00 24.02 O In your case all element names are moved towards left and B value of ZN is in incorrect position also. ATOM 1893 O UNK A 258 -8.934 52.268 49.467 0.00 66.53 O ATOM 1894 ZN+2 ZN B 1 -10.456 38.580 26.267 1.00 57.36ZN+2 ATOM 1895 O HOH C 1 -5.932 42.917 25.589 1.00 24.02 O Regards Garib On 4 Oct 2011, at 15:31, Eleanor Dodson wrote: Can anyone advise me how to get the Zn+2 formfactor from atomsf.lib The input coordinate is given atom type ZN+2 but the formfactor is that for Zn: I changed the atom name to Zn+2 but that made no difference... ATOM 1893 O UNK A 258 -8.934 52.268 49.467 0.00 66.53 O ATOM 1894 ZN+2 ZN B 1 -10.456 38.580 26.267 1.00 57.36ZN+2 ATOM 1895 O HOH C 1 -5.932 42.917 25.589 1.00 24.02 O refmac log says: loop_ _atom_type_symbol _atom_type_scat_Cromer_Mann_a1 _atom_type_scat_Cromer_Mann_b1 _atom_type_scat_Cromer_Mann_a2 _atom_type_scat_Cromer_Mann_b2 _atom_type_scat_Cromer_Mann_a3 _atom_type_scat_Cromer_Mann_b3 _atom_type_scat_Cromer_Mann_a4 _atom_type_scat_Cromer_Mann_b4 _atom_type_scat_Cromer_Mann_c N 12.2126 0.0057 3.1322 9.8933 2.0125 28.9975 1.1663 0.5826 -11.5290 C 2.3100 20.8439 1.0200 10.2075 1.5886 0.5687 0.8650 51.6512 0.2156 O 3.0485 13.2771 2.2868 5.7011 1.5463 0.3239 0.8670 32.9089 0.2508 SE17.0006 2.4098 5.8196 0.2726 3.9731 15.2372 4.3543 43.8163 2.8409 S 6.9053 1.4679 5.2034 22.2151 1.4379 0.2536 1.5863 56.1720 0.8669 ZN14.0743 3.2655 7.0318 0.2333 5.1625 10.3163 2.4100 58.7097 1.3041 Eleanor Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Refinement with twinned data question
Dear Yuri 1) For twin refinement refmac internally changes free reflections so that twin related reflections belong to the same class (twin or working). However it would be good to generate free reflections in higher space group that would include twin and space group symmetries. 2) You should always use original data for refinement. Never use output from refmac for next round of refinement. Output mtz file is representation of model. regards Garib On 2 Oct 2011, at 03:27, Yuri wrote: Dear all, I have some questions about the correct protocol for refinement of twinned data (0.46) 1- How do I ensure that my Rfree set is generated properly? 2-I noticed that if I enable intensity based twin refinement, refmac estimates the correct twin fraction law -h,-k,h+l. But for subsequent refinement rounds should I keep using the raw intensity mtz file or the newly generated mtz with PHIC and FOM information? Thank you -- Yuri Pompeu Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Apparent twinning in P 1 21 1
Why do you detwin? It would not be normal procedure if you have twinning. Molecular replacement programs usually do not have much problem with twinned data and refinement programs can deal with them more or less properly. when you detwin then errors are increased (As far as I remember proportional to 1/(1-2alpha), if alpha is 0.46 then errors will increase more than 10 times). Moreover it is very likely that twin and pseudo rotation are close to each other and estimated twin fractions may not be accurate. regards Garib On 29 Sep 2011, at 15:03, Yuri Pompeu wrote: After I ran DETWIN with the estimated 0.46 alpha, my completeness for the detwinned data is now down to 54%!!! Is this normal behavior? (I am guessing yes since the lower symmetry untwinned dat is P1 21 1) Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac and DNA (and now RNA)
Yes, new version of the dictionary uses U. Refmac will read right or left justified residue names, however pdb may use only one of them. There are some backward compatibility code/dictionary elements. For example Ad/Ar are also accepted. However it is encouraged to use new pdb v2.3 residue/atom names. It will make sure that all software use same naming convention and moving between them is smooth. Hopefully we all will have one naming conventions. regards Garib On 12 Sep 2011, at 19:23, Francis E Reyes wrote: Is ' U ' now the standard vs ' U' ? I'm used to right justified letters for RNA residues in the residue field. This is with a recent refinement with refmac 5.6.0117 . And of course this switch in naming convention breaks compatibility with molprobity (which requires right justified letters in the residue field) F On Sep 8, 2011, at 7:35 AM, Ed Pozharski wrote: After switching (finally) to 6.2.0 and therefore to Refmac 5.6.0117 I have found a problem working with DNA that I have not seen with 6.1.13/5.5.0109. Namely, - if I use the pdb file produced by Coot (0.7.pre-1.3470) that seems to output DNA as Ad/Td/Gd/Cd no matter what the input names were, refmac fails with the warning that it found a new monomer. It appears that it stumbles upon the very first thymidine, but in a strange twist it reports the problematic residue having the name DY! - if I use the pdb file previously produced by refmac, which has the A/T/G/C as residue names, it fails too but now complains about the new monomer named T. - the workaround I found is to rename all the thymidines to DT. It is a bit annoying since coot keeps renaming them (well, not refmac/ccp4 problem per se) and I have to rename back (easily scripted task, of course). What is peculiar is that Ad/Gd/Cd don't need to be renamed (does this have anything to do with thymidine being the only one that changes residue name in RNA?). Has anyone else seen this or it's something specific to my setup? Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] refmac and DNA (and now RNA)
Hi Francis Is that newer version of refmac? regards Garib On 12 Sep 2011, at 20:09, Francis E Reyes wrote: Hi Garib Thanks for the quick reply! Refmac however is writing my pdb's as with the residue letter in the centered position. Is the newest pdb requiring centered residue letters for RNA? F On Sep 12, 2011, at 1:05 PM, Garib N Murshudov wrote: Refmac will read right or left justified residue names, however pdb may use only one of them. - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] CCPALC: not enough memory
I have seen these type of cases. They cause problem. They may come from O, from cns or other programs. Origins may even not be known. My solution was that if an atom has coordinates 9998.000 or more then consider them absent (i.e. set occupancies 0). On 6 Sep 2011, at 14:30, Kevin Cowtan wrote: OK, here's the solution (also to BB in case anyone else has this problem). The PDB file appears to come from CNS. The coordinate file contains the following atoms: ATOM 2244 CG LEU A 452.000.000.000 1.00 0.00 A ATOM 2245 CD1 LEU A 452.000.000.000 1.00 0.00 A ATOM 2246 CD2 LEU A 452.000.000.000 1.00 0.00 A These coordinates are no doubt dummy values of some sort. But, interpreted as coordinates, they are so far from the rest of the model that attempting to build a mask around them and the rest of the model would take a vast file. The PDB file header contains the following lines generated by CNS: REMARK unknown coordinates for atom: ALEU 452 CG REMARK unknown coordinates for atom: ALEU 452 CD1 REMARK unknown coordinates for atom: ALEU 452 CD2 That suggests to me that the problem may have been with the file input to CNS. You'd have to look at your input file to work out exactly what is going on. In the meantime, deleting these atoms or autofitting the sidechain in coot ought to fix it. Hope that solves it, Kevin On 09/06/2011 09:56 AM, Ingo P. Korndoerfer wrote: hello, it seems i am recently getting the above error quite a lot in mapmask. i have searched for a solution, but only found the problem mentioned a few times with no precise solution indicated. would there be a way to find out HOW MUCH memory mapmask is hoping for ? i do have 2 Gb of memory and 3 Gb of swap, and limit says cputime unlimited filesize unlimited datasize unlimited stacksize8192 kbytes coredumpsize 0 kbytes memoryuseunlimited vmemoryuse unlimited descriptors 1024 memorylocked 64 kbytes maxproc unlimited mapmask itself dies in the middle of running and without error message to its log file. thanks ingo Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] twinning in hexagonal system
Hello
Space groups with point groups 622 and 432 merohedral twinning is not possible
(they are the highest groups possible for proteins).
If you could merge in 622 it means that Rmerge was very small. It is very
likely that point group is either 622 or 6 with very strong rotational symmetry
that is perpendicular to 6 fold axis. In these cases H test (sfcheck based on
this) and N(z) test (truncate is based on this) will overestimate (H) or
underestimate (N(z)) if twin is present. At the moment better test for these
cases seems to be L-test (that is in ctruncate I think).
Solving structure using molecular replacement in the presence of twinning does
not seem to be a problem (unless data are very noisy and/or model is too far).
Pointless gives very good idea about true space group.
I would solve in P6_{x} space groups and then run zanuda (from ysbl server) to
correct space group.
I hope it helps
regards
Garib
On 30 Aug 2011, at 20:31, john peter wrote:
Hello All,
This is regarding twinning in a data set.
I collected a native data set to resolution, 1.8 A. I used XDS suite
to process and scale the data set. It scaled well in P622 and I found
systematic absence (l=6n present).
Hence thought the space group may be P6122/P6522. SFCHECK did not
show any twinning and also it did not detect pseudo-translation. Twin
test in http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin
Detector: Padilla-Yeates Algorithm ) showed perfect twinning.
Scaled in P61/65 and SFCHECK reported twinning fraction 0.272 no
pseudo-translation.
http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin Detector:
Padilla-Yeates Algorithm ) showed perfect twinning.
Another server from ucla http://nihserver.mbi.ucla.edu/Twinning/
showed partial twinning with twin fraction 0.23 as follows.
2 along a, b, a*, b*
No. Twin Law Related Reflections = 18033 (pairs)
No. Twin Law Pairs Considered = 9016
H = 0.266149
H2 = 0.095303
Twin Fraction = 0.233249 +/- 0.000602
(SHELXL Commands: TWIN 1 0 0 -1 -1 0 0 0 -1 and BASF 0.233249)
In P61/65, I got the following matthews-coeffs
mol/asym Matthews Coeff %solvent P(1.73) P(tot)
_
19.7587.39 .00 .00
24.8874.79 .00 .01
33.2562.18 .06 .14
4 2.4449.57 .57 .63
5 1.9536.97 .36 .21
6 1.6324.36 .00 .00
7 1.3911.75 .00 .00
_
May I ask what could be the real twin fraction and what is the
likelihood of solving the structure by molecular replacement by models
with 25 % sequence identity and 30 % sequence similarity.
Thank you so much for reading this mail during your busy hours and
all suggestions, comments would be gratefully welcome appreciated.
thank you ccp4 mailing list.
John
Garib N Murshudov
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk
Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] number of cycles in refmac
Hello Tim It was in one or two versions and I did not get consistent results. However code is there and I can activate it if you want. If you know what criteria you would like to use I can code that also. In some cases it happens that R/Rfree go up and then they start coming down. It may be case when starting model has not very nice geometry. We are minimising total function. And there are several hidden parameters in the total function (weights between Xray and geometry etc), they may cause problems. Cheers Garib On 25 Aug 2011, at 10:32, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Ian, I dare say that the goal is to get phases which match as good as possible with what is inside the crystal. If this coincides with maximising the likelihood, why don't we run refinement until the LL stabilises? @Garib: I have seen runs where Refmac actually does stop prematurely, i.e. before the number of cycles set in the input script. It seems that it stops when LL does not drop between two cycles, but according to Ian this would be the point to reach. My question: does this indeed provide the best interpretable map (be it cheating or not)? Cheers, Tim On 08/24/2011 05:36 PM, Ian Tickle wrote: Hi Tim The answer is a definite NO, the goal of refinement is to maximise the likelihood from the working set and the restraints. It is definitely not to optimise Rfree or LLfree. The correct value of the latter is whatever you get at convergence of refinement, i.e. at maximum of the likelihood, anything else is 'cheating'. Cheers -- Ian On Wed, Aug 24, 2011 at 4:24 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear all, especially at the beginning of model building and/or at low resolution both Rfree and -LL free as reported in the refmac logfile show a minimum at a some cycle before rising again. I am certainly not the only one tempted to first run refmac with a large number of refinement cycles, determine that minimum and rerun refmac with ncyc set to that minimum. Of course I want the resulting model and phases/map to be as close to the what's in the crystal as possible in order to facilitate model building. Is it therefore good practice to interrupt refmac wherever it finds a minimum (if so, the minimum w.r.t. which number reported in the log-file)? Thanks for everyone's opinion and experience, Tim - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOVgiBUxlJ7aRr7hoRAq+dAKDj2B6iUMD7C4uu8UMznTlKoclYzACdF8nP Q6DmIFGPcfoP6xbJRwooWWI= =7r5T -END PGP SIGNATURE- Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] number of cycles in refmac
Dear Tim At the moment there is no option to stop refmac prematurely. I can add if it is necessary. I can only give my experience. After molecular replacement before running ARP/wARP or buccaneer I usually run 60-100 cycles of refinement with jelly body with sigma set to 0.01. Then automatic model building works better. I have seen this behaviour in several cases (if there are more than one copy I also would use local ncs restraints). Using jelly body slows down convergence but shifts seem to make more sense. regards Garib On 24 Aug 2011, at 17:24, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear all, especially at the beginning of model building and/or at low resolution both Rfree and -LL free as reported in the refmac logfile show a minimum at a some cycle before rising again. I am certainly not the only one tempted to first run refmac with a large number of refinement cycles, determine that minimum and rerun refmac with ncyc set to that minimum. Of course I want the resulting model and phases/map to be as close to the what's in the crystal as possible in order to facilitate model building. Is it therefore good practice to interrupt refmac wherever it finds a minimum (if so, the minimum w.r.t. which number reported in the log-file)? Thanks for everyone's opinion and experience, Tim - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOVReTUxlJ7aRr7hoRAqyzAKCZpMJPVSQJTDEoWGxZEymwvqfFcACeMNLL rvIDPlXiL5HQmoNm7yrTt6k= =UnKT -END PGP SIGNATURE- Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Another paper structure retracted
Dear all Does anybody have the list (pdb as well as structure factors) of all retracted structures? regards Garib On 10 Aug 2011, at 22:01, David Schuller wrote: Time to fuel up the gossip engines for the approaching weekend: http://www.sciencedirect.com/science/article/pii/S096921260800186X RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer Gβ1γ2 Structure, Volume 16, Issue 7, 9 July 2008, Pages 1086-1094 Structure 2QNS withdrawn. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Mixed Iso/Aniso in refmac5
Thank you for reminding me. There is a way to define residue ranges for mixed refinement. I think I did it by Boaz's request. Keyword should be something like (there are ways of selecting particular atoms of residues, but I am not sure I have tested that thoroughly): brefine mixed anisou residues from resnumber chain to resnumber chain atoms list of atoms Full description of the keywords for mixed refinement is in this document (anisotropic refinement section): http://www.ysbl.york.ac.uk/refmac/data/refmac_news.html Thus option should be available from the latest ccp4 (but you need to use keyword, there is no buttons for this on the interface yet) Please let me know if it does not do what it is designed for. Regards Garib On 5 Aug 2011, at 11:51, Boaz Shaanan wrote: I think Garib has looked into this and introduced an option to specify residues range for iso/aniso B refinement in one of the latest refmac versions. He'll probably jump into the thread soon to clarify this option. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ethan Merritt [merr...@u.washington.edu] Sent: Thursday, August 04, 2011 6:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Mixed Iso/Aniso in refmac5 On Thursday, 04 August 2011, Yuri Pompeu wrote: Hello everyone, How does refmac5 pick atoms for B-factor refinement, particularly with the mixed option enabled? In the MIX option, it simply keeps the atom treatment as it is given in the input file. Atoms with an ANISOU record are refined anisotropically, the remaining atoms are not. In the case of modeling anisotropy using TLS, the atom selection is done in a separate file *.tls or *.tlsin. Ethan I dont see a place for entering a manual selection, eg resname FMN... Thank you Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] hello
Dear AfshanMake sure that group name for CME is peptide (or L-peptide). In the new version CME is peptide. I am not sure it was the case in older versions. I attach CME as a peptide. You can add this into your dictionary.Then CME can become part of a peptde. It would also be good to remove all the link records in the beginning of refmac.RegardsGarib CME.cif Description: Binary data On 21 Jul 2011, at 16:13, Afshan Begum wrote:Dear all,I have facing one problem during the refinement of my protein . Actually in my protein there are some modified amino acids are present like Cystein is modified into CME which i can get easily from monomer libraray in coot . but after refinement in Pdb text file indicated some gaps while in the structures there are no gap in between these amino acids so if any one suggest me what to do. I would appreciate your kind suggestions.LINKR GLU A 142 LEU A 144 gap LINKR SER A 328 GLY A 330 gap LINKR LEU A 138 GLU A 140 gap LINKR GLU A 126 ASP A 130 gap LINKR SER A 246 GLY A 248 gap Many thanks for your timeBest regardsAfshan Garib N MurshudovStructural Studies DivisionMRC Laboratory of Molecular BiologyHills RoadCambridgeCB2 0QH UKEmail:garib@mrc-lmb.cam.ac.ukWebhttp://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Refmac5: put restraint on only peptide bonds
External distance restraints have type type 0 - replace what is generated by refmac type 1 - use if there is no restraint for this type. Only one restraint per atom pair type 2 - there may be many restraints per atom pair. Refmac will choose the restraint that is the closest to current interatomic distance I hope it answers to you question. Cheers Garib On 23 Jun 2011, at 14:21, Ed Pozharski wrote: On Thu, 2011-06-23 at 16:49 +0900, Masaki UNNO wrote: Dear all Is it possible to put restraint on only peptide bonds? I would like to put restraint on O-N bond lengths, Ca-O-N angles, and O=O-N angles. Could you teach me how to do it, if possible? Masaki UNNO, Ph.D. One could create a monomer library with a set of alternative amino acids (you'd rename them in the PDB-file too). Then, modeling a library after the standard one, you assign alternative atom types and have a limited set of defined bonds/angles. This would be quite painful to do though. I wonder what will happen if you run unrestrained refinement yet provide external distance restraints as described in refmac manual? Will the external restraints be ignored? If not, then I'd think this is a better approach. If yes, then maybe you can do the trick by using very high weight matrix parameter (approaching the unrestrained refinement) and correspondingly high external weight scale value. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Problems in refinement
Dear Petr Newer version of refmac is 5.6 and it should be available from ccp4 soon. Meanwhile you can try this version from this website http://www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.6_linux.tar.gz There are versions for mac etc also. regards and I hope you will sort it out soon Garib On 27 May 2011, at 09:57, Petr Kolenko wrote: Dear colleagues, regarding Q2: I do not use TLS parameters, the space group is P1, and yesterday I tried to refine the structure with anisotropic ADP (60,000 reflections against 50,000 parameters) - no positive maximums. Then I used the anisotropic model as input and refined isotropically, the maximums are there again. I use refmac5.5.0100, is it really too late version? Seems to be current version according the websites. Many thanks. Petr 2011/5/27 Jan Dohnalek dohnalek...@gmail.com: In the last months we have seen different versions of Refmac give different maps when displayed in Coot, i.e. one version giving nicer agreement and no difference peaks in some difficult areas and another version resulting in sharp differences where it was hard to build the protein. We did not investigate much further as most of the time use of two or three versions gave us a good picture of what's going on ..probably a feature which would disappear with newer versions anyway. What's the version of Refmac you use, Petr? Jan Dohnalek On Thu, May 26, 2011 at 12:11 PM, Petr Kolenko kole...@imc.cas.cz wrote: Dear colleagues, I have two problems in two structure refinements using REFMAC5. 1) 1.8A resolution, zinc in the active site. Refinement using work reflections - ADP for Zinc was about 14. Final refinement including all reflections increase ADP to 20 or even higher values - followed by very high positive difference density in position of the zinc. I have tried also PHENIX, the same thing. I changed ADP manually to 14 and only calculated maps (no refinement) look good. May I deposit the structure using manually fixed ADP according to the best agreement to the observed and difference electron density? By the way, it is clear that this is zinc. 2) 1.9A resolution, about 600AA, all of them OK in electron density. But, somehow, about ten atoms give very strong positive electron density suggesting they are not taken into account in refinement. On the other hand, ADPs are reasonable and seem to be refined. All of these atoms are fully occupied. I tried to omit whole residues and build them again, but the maxima appeared again. Using of PHENIX resulted in no difference electron density for these atoms. I have also tried to take PHENIX output to REFMAC, but the maxima are there again. It is always one or two atoms from the same residues - sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but still on the same five residues. Does anyone have any idea how to solve this problem? Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410 -- Petr Kolenko kole...@imc.cas.cz http://kolda.webz.cz
Re: [ccp4bb] Problems in refinement
If you are using TLS refinement the please check TLS definitions.It may be that atoms for which you have positive density are not in TLS definitions. Try to use without TLS. regards Garib On 26 May 2011, at 11:11, Petr Kolenko wrote: Dear colleagues, I have two problems in two structure refinements using REFMAC5. 1) 1.8A resolution, zinc in the active site. Refinement using work reflections - ADP for Zinc was about 14. Final refinement including all reflections increase ADP to 20 or even higher values - followed by very high positive difference density in position of the zinc. I have tried also PHENIX, the same thing. I changed ADP manually to 14 and only calculated maps (no refinement) look good. May I deposit the structure using manually fixed ADP according to the best agreement to the observed and difference electron density? By the way, it is clear that this is zinc. 2) 1.9A resolution, about 600AA, all of them OK in electron density. But, somehow, about ten atoms give very strong positive electron density suggesting they are not taken into account in refinement. On the other hand, ADPs are reasonable and seem to be refined. All of these atoms are fully occupied. I tried to omit whole residues and build them again, but the maxima appeared again. Using of PHENIX resulted in no difference electron density for these atoms. I have also tried to take PHENIX output to REFMAC, but the maxima are there again. It is always one or two atoms from the same residues - sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but still on the same five residues. Does anyone have any idea how to solve this problem? Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] do we have to exclude Rfree columns when generating the real space density maps?
It should be remembered that refining in real space is equivalent to refinement in the reciprocal space (through Parseval's theorem). If you want to do consistent refinement then you need to use exactly same reflections for free and working set. If you do not use the same set of reflections for real and reciprocal space refinements then you may get very interesting results. Garib On 23 May 2011, at 21:17, Hailiang Zhang wrote: Thanks Nat! I am not doing real space refinement. Actually I am only using the maps for manual model building/adjustments. In this case, if some Rfree reflections have strong scattering intensities, removing them may lead to featureless density maps. However, if we just leave them in, do you think we may have the so-called model-bias issue? Hailiang On Mon, May 23, 2011 at 1:02 PM, Hailiang Zhang zhan...@umbc.edu wrote: I have a preliminary question. I understand Rfree reflection sets are never used during automatic refinement, but, when generating the real space density maps, do we have to exclude Rfree columns? Any references will also be greatly appreciated! If you are going to run overall real-space refinement on the structure, you should absolutely exclude the test set reflections from the map. If you are only going to run local refinement of small parts of the model in Coot or equivalent, it's debatable - in practice, I think most people/programs leave them in. -Nat
Re: [ccp4bb] do we have to exclude Rfree columns when generating the real space density maps?
It does not matter. By fitting manually you are doing manual minimisation. The same treatment is applied. You are trying to optimise fit of the model into the electron density. I did these tests few years back and results were as expected. Independent on minimisation tools (manual, automatic, partial, full: real space, reciprocal space) exactly same model bias is added. As I said the reason is Parseval's theorem (with some adjustments in case when you are using maximum likelihood refinement) Garib On 23 May 2011, at 21:45, Hailiang Zhang wrote: Thanks Garib, but my task was not real space refinement (just manual model building/adjustment). Following is my previous post. Thanks! I am not doing real space refinement. Actually I am only using the maps for manual model building/adjustments. In this case, if some Rfree reflections have strong scattering intensities, removing them may lead to featureless density maps. However, if we just leave them in, do you think we may have the so-called model-bias issue? Hailiang It should be remembered that refining in real space is equivalent to refinement in the reciprocal space (through Parseval's theorem). If you want to do consistent refinement then you need to use exactly same reflections for free and working set. If you do not use the same set of reflections for real and reciprocal space refinements then you may get very interesting results. Garib On 23 May 2011, at 21:17, Hailiang Zhang wrote: Thanks Nat! I am not doing real space refinement. Actually I am only using the maps for manual model building/adjustments. In this case, if some Rfree reflections have strong scattering intensities, removing them may lead to featureless density maps. However, if we just leave them in, do you think we may have the so-called model-bias issue? Hailiang On Mon, May 23, 2011 at 1:02 PM, Hailiang Zhang zhan...@umbc.edu wrote: I have a preliminary question. I understand Rfree reflection sets are never used during automatic refinement, but, when generating the real space density maps, do we have to exclude Rfree columns? Any references will also be greatly appreciated! If you are going to run overall real-space refinement on the structure, you should absolutely exclude the test set reflections from the map. If you are only going to run local refinement of small parts of the model in Coot or equivalent, it's debatable - in practice, I think most people/programs leave them in. -Nat
Re: [ccp4bb] do we have to exclude Rfree columns when generating the real space density maps?
Dear Keitaro As far as I know different program behave differently. REFMAC by default replaces structure factors of the excluded reflections with their expected values (as a first approximation) that is equal to DFc, where D reflects errors in coordinates. It seems to be a balance between avoiding model bias in map calculations (due to use of FC) and bias in Rfree (due to use of free reflections in minimisation) and noise introduction due to missing reflections. As far as I recall coot uses map coefficients produced by the refinement programs. In case of refmac it uses FWT, PHWT (that has free reflection replaced with their expected values) and DELFWT PHDELWT (that does not have free reflections). In refmac (I am sure in phenix also) there are keywords to turn this option on/off. I hope it helps. regards Garib On 24 May 2011, at 01:02, Keitaro Yamashita wrote: Dear all, I'm very interested in this topic. I have a question about the default behaviors in output reflection files of each refinement softwares. Are test set reflections excluded from the columns for calculating electron density maps? I found in phenix.refine documentation the option electron_density_maps.exclude_free_r_reflections was equal to False by default. (Does this option affect real space refinement in phenix.refine?) And I don't think Coot excludes test set reflections when opening MTZ file... because there's no option to specify the flag number, right? Thanks in advance, Keitaro 2011/5/24 Pavel Afonine pafon...@gmail.com: Hailiang, r-free reflections should not participate in refinement, regardless whether it is real or reciprocal space one, done with machine driven minimizers or your hands moving atoms in Coot. Period. The issue of correcting the map appearance for missing data (resolution or completeness) is relevant but different. Removing the data is noticeable, but most of the time putting aside test set is not critical for map appearance (given that reflections are selected randomly and do not exceed a reasonable fraction of available data); and when it is critical, the cross validation should be done differently anyway. So the answer to your question is: every time you compute a map not just to enjoy its appearance but to use it to improve your model, do not include test flagged reflections into it. Pavel. On Mon, May 23, 2011 at 1:02 PM, Hailiang Zhang zhan...@umbc.edu wrote: Hi, I have a preliminary question. I understand Rfree reflection sets are never used during automatic refinement, but, when generating the real space density maps, do we have to exclude Rfree columns? Any references will also be greatly appreciated! Best Regards, Hailiang
Re: [ccp4bb] problem with LIBCHECK
Dear Geoff Perhaps you could try jligand available from: http://www.ysbl.york.ac.uk/mxstat/JLigand/index.html There are few tutorials how to deal with ligands and links. I hope it helps Cheers Garib On 20 May 2011, at 21:59, Geoffrey Feld wrote: Greetings fellow Crystallographers, I'm working on a structure at 1.8-A resolution that contains an acetone crosslink between 2 cysteines (crosslink was incorporated by adding 1,3-dichloroacetone). I figured that the easiest way to model this is to mutate one of the cysteines to S-acetonylcysteine (CSA in the PDB) and then link it to the other cys. I've seen how to do this in COOT using the mutate-by-overlap function; however, CSA is of course not in the CCP4 library that is installed on our system. I've built restraints for CSA using phenix.elbow and tried importing the residue into COOT that way, still to no avail. So I figured the way to go now is to import the cif file directly into the LIBCHECK library in our system and then I should (in theory) be able to use mutate-by-overlap to place the residue. However, this is where I'm stuck. I can't seem to figure out the notation for importing the cif file into LIBCHECK. I tried using FILE_CIF CSA.cif and I get an error reading title of input file. What am I doing wrong? Is there another approach I should consider? Any help or advice would be greatly appreciated. Cheers, Geoff -- Geoffrey K. Feld Department of Chemistry 492 Stanley Hall University of California, Berkeley Vigilia pretium libertatis
Re: [ccp4bb] Maps after refmac twin refinement
Dear Mary It should not be unless you ask specifically using one of the following keywords mapc sharpen # automatically define sharpening parameters mapc sharpen bvalue # use this bvalue for sharpening with automatic regularisation parameter mapc sharpen alpha value # sharpen with regularisation parameter alpha. Values around 0.1 would be fine (I think) regards Garib On 21 Mar 2011, at 15:03, Mary Ho wrote: Does anyone know if the maps coefficients after twin refinement in refmac are b-sharpened? And if so, is the sharpening value output anywhere? Thanks, Mary X. Ho Postdoctoral Associate Arnold Lab Rutgers
Re: [ccp4bb] Generating a PDB file for a known ligand
It seems that there is a mismatch between dictionary and libcheck versions. Could you please check 1) library vi $CLIBD_MON/a/ADN.cif It should have primes like: ADN O2' OOH1 0.000 0.0000.0000.000 ADN HO2' HH 0.000 0.731 -0.600 -0.203 ADN C2' CCH1 0.000 -1.179 -0.442 -0.676 ADN H2' HH 0.000 -1.460 -1.449 -0.340 2) library version: vi $CLIBD_MON/list/mon_lib_list.cif the version should be something more than 5.23 (I think) 3) libcheck version libcheck the version should be something more than 5.1 If one of these is different then there will be inconsistency and most probably failure. I thought new version of ccp4 has all the latest versions. I may be wrong. regards Garib P.S. When you run libcheck it prints out the versions of the program and the dictionary. On 15 Mar 2011, at 14:43, Phil Evans wrote: Andrew The default libraries installed at LMB are for the latest refmac/libcheck, which use PDB v3 names for nucleotides (ie with primes), for both refmac and coot The older libraries are around somewhere I get extremely confused by this sort of thing Phil Hi Ian, This is bizarre, we also have 6.1.13 installed here, but in my ADN.cif (dated Oct 29 2008) the atom names have primes, but are surrounded by quotes which I think allows a mechanism for them to be converted to a * if you have the appropriate changes listed in your (personal) library. I don't understand why these differ (unless Phil change the ADN.cif entry here for some reason). I think we need a comment for the library people ! Cheers Andrew On 15 Mar 2011, at 14:23, Ian Clifton wrote: On 15/03/11 12:57, A Leslie wrote: … I then try using LIBCHECK standalone to get the PDB file. I get the same error if I use the FILE_CIF input keyword and give it the filename for ADN.cif, no surprise, as this is (I assume) what COOT does. I then copy over ADN.cif to my local directory, run LIBCHECK again and use the FILE_L keyword to specify the (local) ADN.cif file. I then get the error: ERR: item _chem_comp_tree.atom_id :O2' not found in the atom list MON,BLOCK :ADN data_comp_ADN suggesting that there is an error in ADN.cif ! I can’t reproduce this (CCP4-6.1.13), but I’m immediately suspicious of the apostrophe (U+0027) character in the error message. I’m not a DNA dabbler, so I can’t help much, but I bet the problem is due to “primed” atom names being replaced by stars, or something else. In my CCP4 distribution, the atom names are starred in the dictionary file for ADN. In official PDB files, they’re primed I think. -- Best wishes, Ian.
Re: [ccp4bb] Density sharpening with Truncate?
I would not sharpen structure factors before refinement. It may cause problems with B value refinement (a lot of B values may stuck around 2 or minimum B). One must remember that not all atoms in crystal have same Bvalue. There is a distribution of Bvalues. However maps can be sharpened after refinement. It can be done directly in coot (I hope this version of coot is now widely available). Or if you are using refmac for refinement you can use: mapc sharpen # regularised map sharpening. Bvalues and regularisation parameters are calculated automatically or mapc sharpen Bvalue # regularised map sharpening with specified Bvalue or mapc sharpen Bvalue mapc sharpen alpha value=0.1 # regularisation paramater. alpha=0 is simple sharpening. I am sure other programs have similar options. (I know CNS has and it has been used successfully by many people) regards Garib P.S. These options available from refmac v5.6 available from; www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac5.6_linux.tar.gz On 25 Feb 2011, at 23:57, Dima Klenchin wrote: At 05:39 PM 2/25/2011, Pete Meyer wrote: Or could anyone suggest a program that would be of help? CAD scaling with a scale factor of 1.0 and negative B-factor (isotropic or anisotropic) should do the trick. I haven't had much luck with density sharpening (at least at ~4-5 Angstroms), but others have apparently had some success with it. Alternatively, CCP4i task Run FFT does the job: 1. Take MTZ from Refmac output 2. Run FFT to create simple map with SigmaA-weighted phases (i.e., PHWT label). 3. In Infrequently used options, Apply B-factor scaling to F1, specify negative B-factor scaling value, usually within -10 to -50. - Dima
Re: [ccp4bb] very low R factor for twin refinement
Just a warning: When doing twin refinement (and in general) you should always use original data for refinement. After refinement data may be different (in case of twin de-twinned). I will have a look as soon as I have some time. regards Garib On 10 Feb 2011, at 22:06, Patskovsky Yury wrote: Thanks, Garib It might be the case. As a matter of fact, you are welcome to look at the original data here http://www.rcsb.org/pdb/explore/explore.do?structureId=2GL5 The data turned out to be twinned ( I also have other datasets - all with certain degree of twinning) and now I am trying to re-refine and then re-submit the more correct structure to the PDB. Yury On Thu, 10 Feb 2011, Garib N Murshudov wrote: Maximum theoretical drop R/Rfree for perfect twin from 30% is around 25% (i.e. it could go down to 5%). However it could only happen only if twinning is perfect and there is no pseudo rotation parallel to twin operator. Hypothetical case it can happen if you have refined one crystal structure at sufficiently high resolution till (almost convergence) and another crystal is twinned but otherwise perfectly isomorphous to the first crystal and you take coordinates from the first crystal and refine against the second crystal. regards Garib On 10 Feb 2011, at 20:14, Patskovsky Yury wrote: Dear all, Twin refinement has yielded Rwork/Rfree values of about 0.10/0.12 for a nice quality 1.8A dataset (Rmerge 6%, space group I4, twin fractions 0.6/04) and almost the same R/Rfree (0.095/0.115) for another 1.5A nice quality data set (Rmerge 6%, space group I4, twin fractions 0.74/0.26). Refinement of untwinned data resulted in Rfree of ~32% and ~22% respectively. REFMAC and PHENIX both have produced the same results and almost identical R factors, which are suspiciously VERY LOW for this resolution of data. Twin refinement in REFMAC has produced exceptional quality maps even for 1.8A data (they look rather like 1.2A maps) - I can not tell the same for PHENIX - maps were looking worse (may be someone has a better idea why). Normally twin refinement results in lowering R-factors - say, the drop in R from 30% (without twin refinement) to 20% (with twin refinement) would be considered normal, however we can see the drop from 32% to 12%. I wonder if anyone else has experienced similar problems and what would be the most reasonable explanation for that. Thank you Yury Yury Patskovsky, Ph.D. Associate, Dept of Biochemistry Albert Einstein College of Medicine 1300 Morris Park Ave Bronx, NY 10461 phone 718-430-2745 yu...@medusa.vioc.aecom.yu.edu
Re: [ccp4bb] very low R factor for twin refinement
Dear Yury I looked at this data and such drop of R/Rfree when you switch from non-twin to twin in the presence of twinning is expected. When you said that electron density is too good I was afraid that there is strong bias. But at least for the case from the pdb (2gl5) you can see a lot of features that are not in the model (double conformations, waters, end even some unexplained ligands etc). In general, maps, especially unexplained parts are best indicators of bias towards the errors in the model. Regards Garib On 10 Feb 2011, at 22:06, Patskovsky Yury wrote: Thanks, Garib It might be the case. As a matter of fact, you are welcome to look at the original data here http://www.rcsb.org/pdb/explore/explore.do?structureId=2GL5 The data turned out to be twinned ( I also have other datasets - all with certain degree of twinning) and now I am trying to re-refine and then re-submit the more correct structure to the PDB. Yury On Thu, 10 Feb 2011, Garib N Murshudov wrote: Maximum theoretical drop R/Rfree for perfect twin from 30% is around 25% (i.e. it could go down to 5%). However it could only happen only if twinning is perfect and there is no pseudo rotation parallel to twin operator. Hypothetical case it can happen if you have refined one crystal structure at sufficiently high resolution till (almost convergence) and another crystal is twinned but otherwise perfectly isomorphous to the first crystal and you take coordinates from the first crystal and refine against the second crystal. regards Garib On 10 Feb 2011, at 20:14, Patskovsky Yury wrote: Dear all, Twin refinement has yielded Rwork/Rfree values of about 0.10/0.12 for a nice quality 1.8A dataset (Rmerge 6%, space group I4, twin fractions 0.6/04) and almost the same R/Rfree (0.095/0.115) for another 1.5A nice quality data set (Rmerge 6%, space group I4, twin fractions 0.74/0.26). Refinement of untwinned data resulted in Rfree of ~32% and ~22% respectively. REFMAC and PHENIX both have produced the same results and almost identical R factors, which are suspiciously VERY LOW for this resolution of data. Twin refinement in REFMAC has produced exceptional quality maps even for 1.8A data (they look rather like 1.2A maps) - I can not tell the same for PHENIX - maps were looking worse (may be someone has a better idea why). Normally twin refinement results in lowering R-factors - say, the drop in R from 30% (without twin refinement) to 20% (with twin refinement) would be considered normal, however we can see the drop from 32% to 12%. I wonder if anyone else has experienced similar problems and what would be the most reasonable explanation for that. Thank you Yury Yury Patskovsky, Ph.D. Associate, Dept of Biochemistry Albert Einstein College of Medicine 1300 Morris Park Ave Bronx, NY 10461 phone 718-430-2745 yu...@medusa.vioc.aecom.yu.edu
Re: [ccp4bb] Let's talk pseudotranslational symmetry (or maybe it's bad data).
molrep can deal with some of the PST cases. Garib On 9 Feb 2011, at 22:27, Phil Jeffrey wrote: Is there a program that does ? I was under the impression that they were all equally good/bad at this, because any solution that agrees with the PTS has quite a high score and any solution that doesn't has a low score, irrespective of the correctness of the placement of the molecules. In one case that ritually defeats me with quite strong pseudo-centering, this seems to be true for heavy atom searches also. Phil Jeffrey Princeton On 2/9/11 5:08 PM, Jon Schuermann wrote: I would NOT use Phaser for MR with PTS present. It doesn't handle it correctly yet, since the likelihood targets don't account for PTS. Others may be able to explain it better.
Re: [ccp4bb] A small bug in the CCP4 dictionary?
It indeed seems to be case. Thank you for pointing this out. I have change in the version I have and it will be available soon. reagrds Garib On 20 Jan 2011, at 14:26, Thomas Womack wrote: The restraint dictionary for hydrogenated tryptophan, lib/data/monomers/t/TRP.cif, lists a 15-atom plane for the sidechain, omitting the atom HZ2. Unless this is an exciting result derived from neutron diffraction experiments, would it be possible to fix the dictionary? Yours sincerely, Thomas Womack (Global Phasing)
Re: [ccp4bb] Refmac: sidechain bond breaks
Dear Marcus The most likely reason is that geometry is a bit loose. You need to tighten it a bit. You can do by decreasing weight using weight matrix option on the interface. You need also check the electron density to make sure that ILE is in electron density. Please let me know if you have any further difficulty. regards Garib On 17 Jan 2011, at 09:28, Marcus Fislage wrote: Dear all, I might excuse myself for the silly question but it is the first time I solve an x-ray structure. After modell building in coot and running of refmac with restrained refinement I have the problem that the pdb output file contains distances between e.g. ILE Cb and Cg that are so long that the Cg is displayed as single atom (distance ~1.64 A instead of 1.5 A). This seems to happen to me for aminoacid sidechains where I added an alternative conformation (especially if the 2nd conformation is oriented not far away from the first one) and if I set the occupancy of sidechains like Glu for Cd and further to zero (Here refmac gives a bond break between the last atom with occupancy 0 and the first with occupancy 1). I can adjust it of course in coot back to normal but after the next refmac run the same happens again. The option card in refmac for geometric restraints I kept untouched. Am I missing to set on an option in refmac, or is there something else going wrong? Thanks a lot for the help Marcus
Re: [ccp4bb] dictionary files of polysaccharide for refmac run
Hi they are in the standard dictionary. To make sure that you have full range of ligand you may take the dictionary from www.ysbl.york.ac.uk/refmac/data/refmac_experimental/refmac_dictionary_v5.23.tar.gz and corresponding refmac www.ysbl.york.ac.uk/refmac/refmac_experimental/refmac5.6_linux.tar.gz The dictionary has almost all sugars, links between sugars, sugar protein links and many more. This version of refmac is consistent with pdb v3 and has around 10500 ligands (all ligands that were deposited to pdb by Nov 2010) regards Garib On 10 Jan 2011, at 23:02, Hailiang Zhang wrote: Hi, I am running refmac on gp120(PDB 3FUS), and wondering whether there are any dictionary files (.cif) that have already been built for the polysaccharide (containing FUL BMA MAN NAG NDG, with NAG linked to ASN). Thanks in advance for any help! Best Regards, Hailiang
Re: [ccp4bb] Refmac twin refinement and the output map
Dear Yu I cannot say about other programs. refmac uses equation in slides No 13-14 of the presentation: http://www.ysbl.york.ac.uk/refmac/Presentations/ Refmac_Erice_workshop.ppt If your crystal is a perfect twin and you have processed data in true space group then refmac will give map for a single crystal. However accuracy of results have never been analysed. Moreover I would be very careful in the beginning of refinement (i.e. immediately after molecular replacement with high R factors). In these cases twin fraction refinement and map coefficients may not be as reliable as they should be. regards Garib On 15 Dec 2010, at 17:25, zhang yu wrote: Dear all, I have a question about twin refinement of Refmac in CPP4. I was told that the refmac (Phenix also?) will generate the detwined map after the refinement with twin. My question is that If my crystal is a perfect hemihedral twin, how can the refmac make a detwined map after the refinement? Thanks -- Yu Zhang HHMI associate Waksman Institute, Rutgers University 190 Frelinghuysen Rd. Piscataway, NJ, 08904
Re: [ccp4bb] Need no clash evaluation among symmetry mates during refinement
At the moment there is no way of telling refmac to ignore clashes between symmetry related atoms. Is there any reason you would want to do that? Perhaps there are other ways of solving the problem you would like to solve. I can add a keyword to force to ignore these clashes if there is sufficiently good argument to do so. If you want to deal with translational disorder (e.g. you have a modulated crystal then there is another way. You just give your molecules and then ask the program to ignore clashes. regards Garib On 8 Dec 2010, at 16:45, Keitaro Yamashita wrote: Dear Ed, These tables were reported by Refmac5: Before refinement, --- Restraint type N restraints Rms Delta Av(Sigma) . VDW repulsions: symmetry: refined_atoms 38 0.232 0.200 VDW repulsions: symmetry: others100 0.393 0.200 HBOND: symmetry: refined_atoms 11 0.229 0.200 HBOND: symmetry: others 1 0.120 0.200 . --- After refinement, VDW repulsions: symmetry: refined_atoms 27 0.190 0.200 VDW repulsions: symmetry: others 76 0.262 0.200 HBOND: symmetry: refined_atoms 13 0.225 0.200 I thought that meant there's vdw repulsion energy among symmetry related atoms, which I want refmac5 to ignore. K. Yamashita 2010/12/9 Ed Pozharski epozh...@umaryland.edu: On Thu, 2010-12-09 at 01:09 +0900, Keitaro Yamashita wrote: When I tried to refine using Refmac5, the output told many vdw repulsions with symmetry mates What do you mean by that? I had a similar situation recently, and there are many records in the log file that say something like this INFO: link is found (not be used) dist= 1.531 ideal_dist= 1.500 ch:AA res: 217 GLN at:C .-ch:FF res: 217 GLN at:CA . Note the not be used part. AFAIU, these clashes are simply reported but ignored in refinement. Perhaps some keyword like monitor none shall turn these warnings off, but other than making your log-files very long it's not a problem. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Need no clash evaluation among symmetry mates during refinement
In refmac you can remove vdw interactions between chains using the following command It is an example: vdwr exclude between chains A B or between resdues: vdwr exclude between residues first residue 123 chain A second residue 155 chain B Regards Garib On 8 Dec 2010, at 16:09, Keitaro Yamashita wrote: Dear all, I'm refining complex structure against X-ray diffraction data with packing disorder. (Some domains overlap with their symmetry mates (4-fold), so their occupancies are set to 0.25) I'd like to know whether refinement programs can exclude any interaction among symmetry mates from geometric term in target function. Can it be done only for specific chains? I was thankfully taught phenix.refine could do that with the option pdb_interpretation.custom_nonbonded_symmetry_exclusion in phenixbb. I'd very much like to know whether Refmac5, CNS or other programs can do that. (When I tried to refine using Refmac5, the output told many vdw repulsions with symmetry mates.) Thank you very much in advance, K. Yamashita
Re: [ccp4bb] Need no clash evaluation among symmetry mates during refinement
Dear Yamashita Refmac indeed does not apply vdw repulsions (antibumping restraints) if the sum of occupancies is less than 1 and atoms do not belong to the same residue with the same alt code. It means that if you have two or more molecules each with occupancy less than 0.5 then there will be no intramolecular antibumping restraints also. It is not what you would like. In these case you may want to apply antibumping restraints within molecule. For this reason using keyword (if you have three copies and chain names are A B and C) vdwrepulsions exclude between chains A B C would be better. Then the program would apply antibumping restraints within molecule but not between them. Your case: Do you see some sort of modulation of intensities in your images? Something like weak strong intensities along c axis? Or do you see elongated peaks in the images. regards Garib On 9 Dec 2010, at 01:11, Keitaro Yamashita wrote: Dear Garib, Sorry I'm a little confused. As Eleanor said, Is it true that Refmac doesn't apply vdw restraints if the sum of the atoms occupancies is = 1? If you want to deal with translational disorder (e.g. you have a modulated crystal then there is another way. In my data, there are crystallographic 4-fold axis on c-axis (P4 21 2) and pseudo-translation vector (0.1, 0.1, 0.5), which is indicated by native patterson peak with 22% height of the origin. I can see very clear density at certain position around the 4-fold axis. And around the shifted position (+0.1, +0.1, +0.5), there's definitely the same density but they are superposed with their symmetry mates -- I think they are statically (packing) disordered. Is my case translational disorder as you said? If so, how can I solve it? Yours truly, K. Yamashita 2010/12/9 Garib N Murshudov ga...@ysbl.york.ac.uk: In refmac you can remove vdw interactions between chains using the following command It is an example: vdwr exclude between chains A B or between resdues: vdwr exclude between residues first residue 123 chain A second residue 155 chain B Regards Garib On 8 Dec 2010, at 16:09, Keitaro Yamashita wrote: Dear all, I'm refining complex structure against X-ray diffraction data with packing disorder. (Some domains overlap with their symmetry mates (4-fold), so their occupancies are set to 0.25) I'd like to know whether refinement programs can exclude any interaction among symmetry mates from geometric term in target function. Can it be done only for specific chains? I was thankfully taught phenix.refine could do that with the option pdb_interpretation.custom_nonbonded_symmetry_exclusion in phenixbb. I'd very much like to know whether Refmac5, CNS or other programs can do that. (When I tried to refine using Refmac5, the output told many vdw repulsions with symmetry mates.) Thank you very much in advance, K. Yamashita
Re: [ccp4bb] How to tighten the linkage between ASN NAG
If you will replace link line with the following LINKRC1 NAG A1003 ND2 ASN A 611NAG-ASN Then it should be read by refmac and by coot. And restraints should be applied properly regards Garib On 2 Dec 2010, at 06:49, Dr. STEPHEN SIN-YIN, CHUI wrote: Dear All, Please see the maps for the ASN residues of the protein, As suggested by colleague, they are NAG which are connected to the N atom of ASN413 ASN611. I put the NAG molecule to those density and try to use the following command line in PDB, LINKRC1 NAG A1003 ND2 ASN A 611 1555 1555 1.45 but after restrained refinement using REFMAC5 (quite latest version), the bond distance between N of ASN and C1 of NAG seems to be off (1.28 A) from the value I set for this restraint. My question is related to the true command of bond restraint for this situation? Also if I want to show a link or line between N atom of the ASN and C1 atom of NAG in COOT, any idea how to do this? many thanks in advance stephen -- Dr. Stephen Sin-Yin Chui Research Assistant Professor, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory) 611-ASN-view1413-ASN-view2
[ccp4bb] jligand
Dear All New version of jligand - version 1.0.0 is now available to download and use. It can be downloaded from http://www.ysbl.york.ac.uk/mxstat/ There are also tutorials how to jligand to create ligand and link descriptions. jligand is a program to create new ligand descriptions. It also can create description of covalent links between ligands as well as ligand proteins. These descriptions are used by refmac5 as well as coot (Paul will correct me if I am wrong) If you have any problems or suggestions please send an email to Andrey (preferable option) or me. With best regards Andrey and Garib
