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Re: [phenixbb] phenixbb Digest, Vol 78, Issue 9
by CPMAS Chen 08 May '12

08 May '12

Thank you all. Actually, as Pavel said there is a easy way to do so, I changed the cif file of the ligand, where I had restraints on the dihedral modified. Message: 3 > Date: Sat, 05 May 2012 13:48:06 -0700 > From: Pavel Afonine <pafonine(a)lbl.gov> > To: phenixbb(a)phenix-online.org > Subject: Re: [phenixbb] how to define a molecule in two rigid_body > Message-ID: <4FA59206.7090207(a)lbl.gov> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Hi Charles, > > I think you are trying to achieve two goals, "keep conformation you > like" and "make sure it fits given density", while one of the two > contradicts to the other. That is either the density you are trying to > fit in your molecule or your expectations about the geometry of the > molecule (or both) are not quite right. > > I think this is how much one can tell given the information you provided. > > I don't think you need to do that sophisticated rigid-body kind of > refinement that you described in your original post. The solution must > be easier. May be if you send me the model and data and tell which part > of the model you think is in trouble then probably I could help more. > > Pavel > > On 5/5/12 10:22 AM, Nathaniel Echols wrote: > > On Sat, May 5, 2012 at 6:39 AM, CPMAS Chen<cpmasmit(a)gmail.com> wrote: > >> I am almost at final stage. but the refinement always shows error > density at > >> the single bond connecting the two rings. I have tried manually change > the > >> orientation of these two rings, but there are two concerns that I > thought > >> why I did not get the perfect fitting. > >> 1. I might not choose the perfect the orientation of these rings, and > these > >> give me a non-perfect fitting, and high b-factor. > >> 2. since phenix always tries to change the conformation a little bit to > fit > >> the density, so this also might cause some errors, or not the > conformation I > >> like. > >> > >> Then, my sense is that, if I can somehow define the two parts of the > >> molecule as two rigid bodies, then phenix only can has free rotation of > >> these two rings, and the fitting might come out good. > > You can define custom planarity restraints for the rings - there isn't > > good documentation for this, but here is what to do: > > > > 1. If you're using the command line, look here in the .eff file: > > > > refinement { > > geometry_restraints { > > edits { > > planarity { > > atom_selection = resseq 1001 and (name C1 or name C2 or name > > C3 or name C4 or name C5 or name C6) > > sigma = 0.1 > > } > > } > > } > > } > > > > (Other parameters omitted for clarify, of course. I'm not sure what > > an appropriate sigma would be - you'll probably have to experiment.) > > > > 2. If you're using the GUI, make sure you have the latest nightly > > build, and on the middle configuration tab, click "Custom geometry > > restraints", which will open a window that includes a tab for creating > > planarity restraints. > > > > Technically, the two planes won't actually be "rigid bodies" (i.e. > > "constrained"), but if you make the planarity restraint sufficiently > > tight there will be very little deviation from ideality. (Just don't > > make it too tight, or you'll confuse the minimizer.) > > > > -Nat > > _______________________________________________ > > phenixbb mailing list > > phenixbb(a)phenix-online.org > > http://phenix-online.org/mailman/listinfo/phenixbb > > > > ------------------------------ > > -- *************************************************** CPMAS Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

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Difficult dataset and refinement--P422? I422?Twinning?
by Zhou, Tongqing (NIH/VRC) [E] 07 May '12

07 May '12

Dear All, I have collected several dataset for an antibody:antigen complex. It apparently indexed to P4 space group with large cell dimensions of 191.7000/191.7000/311.0540. When I processed as P1 and ran Pointless, it suggested P422 as space group. P4212 gives the best MR solution with 6 mol/ASU (18 chains total ). However, ridigbody and subsequent refinement with TLS could not reduce the r and r-free which stayed at ~48%. Tried to go P4, P222...(12 mol/ASU) with twinning, but the refinement failed due to insufficient computer memory (12 GB), it was also impossible to refine in P1 due to the same reason. But for some data sets, HKL2000 also gave I4 space group with c axis reduced to 1/3 of that of P4. With the I4 data (~2.7 A resolution), I422 was the best MR space group (1 mol/ASU), the refinement went down to r=29% and r-free=34%, but won't go down further. Please advise if it's possible to use the data Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4609B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ****************************************************************************** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ******************************************************************

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how to define a molecule in two rigid_body
by CPMAS Chen 05 May '12

05 May '12

Thanks, Ralf. I am almost at final stage. but the refinement always shows error density at the single bond connecting the two rings. I have tried manually change the orientation of these two rings, but there are two concerns that I thought why I did not get the perfect fitting. 1. I might not choose the perfect the orientation of these rings, and these give me a non-perfect fitting, and high b-factor. 2. since phenix always tries to change the conformation a little bit to fit the density, so this also might cause some errors, or not the conformation I like. Then, my sense is that, if I can somehow define the two parts of the molecule as two rigid bodies, then phenix only can has free rotation of these two rings, and the fitting might come out good. Charles %%%%%%%%%%%%%%%%%%%%% University of Pittsburgh School of Medicine Department of Aneshesiology %%%%%%%%%%%%%%%%%%%%%

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how to define a molecule in two rigid_body
by CPMAS Chen 05 May '12

05 May '12

Dear Colleagues, First, I would say sorry to bother your guys at Friday evening. :) I have a molecule which has two rings connected by a single bond. I would like this two rings freely rotate around the single bond, but keep each ring conformation fixed (as there are sidechains connected to each ring). Could you help me do this rigid_body definition, or suggest a better idea to do so in phenix? Regards and Thanks! Charles Chen. %%%%%%%%%%%%%%%%%%%%% University of Pittsburgh School of Medicine Department of Aneshesiology %%%%%%%%%%%%%%%%%%%%%

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geometry weight optimization with mlhl target
by Mooers, Blaine H.M. (HSC) 04 May '12

04 May '12

Dear Phenixbb, How does one optimize geometry when using the mlhl target in refinement with Henderson-Lattman coefficients in the input reflection file? The wxc_scale keyword is not recognized when I used the mlhl target, but it is recognized when I use the ml target by setting main.use_experimental_phases=false. Blaine Blaine Mooers Assistant Professor Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center S.L. Young Biomedical Research Center Rm. 466 Letter address: Shipping address: P.O. Box 26901, BRC 466 975 NE 10th Street, BRC 466 Oklahoma City, OK 73190 Oklahoma City, OK 73104-5419 office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910 e-mail: blaine-mooers(a)ouhsc.edu<mailto:[email protected]> webpage: http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/…

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secondary structure restraints helices/helixes proline
by Morten Groftehauge 04 May '12

04 May '12

Hi guys, How does phenix.refine handle prolines in helices? When I look in high resolution structures it seems that the carbonyl of the residue four positions before the proline (also in the helix) should be more perpendicular than parallel to the helix. This makes sense since there isn't any nitrogen for it to hydrogen bond to. But when I refine my structure it seems as if phenix.refine is trying to push the carbonyl into parallel position. Am I imagining things? It's not the best data and this is a troublesome region. Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University

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Re: [phenixbb] Geometry Restraints - Anisotropic truncation
by Terwilliger, Thomas C 03 May '12

03 May '12

Hi Kendall, Yes, I think you could use this kind of approach to make overall decisions of any kind, including those you suggest. I would not use Rsleep for anything at all, other than calculating a final number. I would use a fixed Rfree set (which could be a subset of the total free set or the whole set) for all such decision making. If a lot of such decisions are made with Rfree...then yes it would be good to have an Rsleep to make sure that everything is ok. All the best, Tom T ________________________________ From: Kendall Nettles [knettles(a)scripps.edu] Sent: Thursday, May 03, 2012 9:05 AM To: Terwilliger, Thomas C; PHENIX user mailing list Subject: Fwd: [phenixbb] Geometry Restraints - Anisotropic truncation Hi Tom, Do you think something like this could be used during refinement to identify the "best" resolution limits? If you have an Rsleep set would Rfree be sufficient for this? I imagine collecting data with a ring of noise and then let the optimal resolution be determined during refinement. My understanding of this is that the modern refinement algorithms can handle some noise in the reflections, but maybe this could be a way to optimize how much signal is needed to contribute in a positive fashion? Kendall

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Re: [phenixbb] Geometry Restraints - Anisotropic truncation
by Leonid Sazanov 03 May '12

03 May '12

Hi, so will we see now the possibility to apply anisotropic resolution limits truncation in Phenix? Despite the fact that a lot of people use UCLA server, there is still no possibility to do this in any major crystallographic software, AFAIK. Phaser does the scaling, but no truncation, and noise sitting in those regions where there is no signal, is a real culprit. Large SIGFs there do not resolve the problem completely. Thanks. >>>>>> Message: 4 Date: Wed, 25 Apr 2012 09:05:57 -0400 From: Mario Sanches <mariosan(a)gmail.com> To: PHENIX user mailing list <phenixbb(a)phenix-online.org> Subject: [phenixbb] Geometry Restraints - UPDATE .......... It turns out that the reason to my nightmares was a highly anisotropic dataset. When I tried to fix geometry related problems in coot, phenix.refine was twisting the geometry back again, probably because it was trying to fit a lot of noise due to the anisotropy. Everything was getting worst, geometry, clashscore, and Ramachandran. Pavel finally corrected it by using this server: http://services.mbi.ucla.edu/anisoscale/ ............ -- Dr. Leonid A. Sazanov Research group leader Medical Research Council Mitochondrial Biology Unit Wellcome Trust / MRC Building Hills Road Cambridge CB2 0XY WEB: www.mrc-mbu.cam.ac.uk Tel: +44-1223-252910 Fax: +44-1223-252915

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Secondary Structure Definitions
by Antony Oliver 03 May '12

03 May '12

Dear all, Is there a phenix jiffy to assign (and write!) secondary structure definitions to a pdb file? I'm currently using MacPyMOL to put together some figures, and I can see that some of the secondary structure elements in the cartoon representation aren't being drawn correctly. Being slightly old-school, I used to use DSSP and the little program dssp2pdb to extract the information and put it into the PDB header. Is there a simpler / easier way to do this? (NB: I'm aware that CCP4MG copes better with SS assignment, but I prefer to use PyMOL in this instance). With thanks, Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oliver(a)sussex.ac.uk<mailto:[email protected]> tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512

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Fwd: Geometry Restraints - Anisotropic truncation
by Kendall Nettles 03 May '12

03 May '12

Hi Tom, Do you think something like this could be used during refinement to identify the "best" resolution limits? If you have an Rsleep set would Rfree be sufficient for this? I imagine collecting data with a ring of noise and then let the optimal resolution be determined during refinement. My understanding of this is that the modern refinement algorithms can handle some noise in the reflections, but maybe this could be a way to optimize how much signal is needed to contribute in a positive fashion? Kendall From: "Terwilliger, Thomas C" <terwilliger(a)lanl.gov<mailto:[email protected]>> Date: May 3, 2012 10:23:29 AM EDT To: PHENIX user mailing list <phenixbb(a)phenix-online.org<mailto:[email protected]>> Subject: Re: [phenixbb] Geometry Restraints - Anisotropic truncation Reply-To: PHENIX user mailing list <phenixbb(a)phenix-online.org<mailto:[email protected]>> Hi Kendall, This could work. You could define a fixed set of test reflections, and never touch these, and never include them in refinement, and always use this fixed set to calculate a free R. Then you could do whatever you want, throw away some work reflections, etc, refine, and evaluate how things are working with the fixed free R set. All the best, Tom T ________________________________________ From: phenixbb-bounces(a)phenix-online.org<mailto:[email protected]> [phenixbb-bounces(a)phenix-online.org<mailto:[email protected]>] on behalf of Kendall Nettles [knettles(a)scripps.edu<mailto:[email protected]>] Sent: Thursday, May 03, 2012 7:05 AM To: PHENIX user mailing list Subject: Re: [phenixbb] Geometry Restraints - Anisotropic truncation Hi Pavel, Could you use a similar approach to figuring out where to cut your data in general? Could you compare the effects of throwing out reflections in different bins, based on I/sigma, for example, and use this to determine what is truly noise? I might predict that as you throw out "noise" reflections you will see a larger drop in Rfree than from throwing out "signal" reflections, which should converge as you approach the "true" resolution. While we don't use I/sigma exclusively, we do to tend towards cutting most of our data sets at the same i/sigma, around 1.5. It would be great if there was a more scientific approach. Best, Kendall On May 3, 2012, at 7:45 AM, Pavel Afonine wrote: Hi Kendall, removing same amount of data randomly gives Rwork/Rfree ~ 30/35%. Pavel On 5/3/12 4:13 AM, Kendall Nettles wrote: Hi Pavel, What happens if you throw out that many reflections that have signal? Can you take out a random set of the same size? Best, Kendall On May 3, 2012, at 2:41 AM, "Pavel Afonine"<pafonine(a)lbl.gov<mailto:[email protected]>> wrote: Hi Kendall, I just did this quick test: calculated R-factors using original and anisotropy-corrected Mike Sawaya's data (*) Original: r_work : 0.3026 r_free : 0.3591 number of reflections: 26944 Truncated: r_work : 0.2640 r_free : 0.3178 number of reflections: 18176 The difference in R-factors is not too surprising given how many reflections was removed (about 33%). Pavel (*) Note, the data available in PDB is anisotropy corrected. The original data set was kindly provided to me by the author. On 5/2/12 5:25 AM, Kendall Nettles wrote: I didnt think the structure was publishable with Rfree of 33% because I was expecting the reviewers to complain. We have tested a number of data sets on the UCLA server and it usually doesn't make much difference. I wouldn't expect truncation alone to change Rfree by 5%, and it usually doesn't. The two times I have seen dramatic impacts on the maps ( and Rfree ), the highly anisotrophic sets showed strong waves of difference density as well, which was fixed by throwing out the noise. We have moved to using loose data cutoffs for most structures, but I do think anisotropic truncation can be helpful in rare cases. Kendall On May 1, 2012, at 3:07 PM, "Dale Tronrud"<det102(a)uoxray.uoregon.edu<mailto:[email protected]>> wrote: While philosophically I see no difference between a spherical resolution cutoff and an elliptical one, a drop in the free R can't be the justification for the switch. A model cannot be made more "publishable" simply by discarding data. We have a whole bunch of empirical guides for judging the quality of this and that in our field. We determine the resolution limit of a data set (and imposing a "limit" is another empirical choice made) based on Rmrg, or Rmes, or Rpim getting too big or I/sigI getting too small and there is no agreement on how "too big/small" is too "too big/small". We then have other empirical guides for judging the quality of the models we produce (e.g. Rwork, Rfree, rmsds of various sorts). Most people seem to recognize that the these criteria need to be applied differently for different resolutions. A lower resolution model is allowed a higher Rfree, for example. Isn't is also true that a model refined to data with a cutoff of I/sigI of 1 would be expected to have a free R higher than a model refined to data with a cutoff of 2? Surely we cannot say that the decrease in free R that results from changing the cutoff criteria from 1 to 2 reflects an improved model. It is the same model after all. Sometimes this shifting application of empirical criteria enhances the adoption of new technology. Certainly the TLS parametrization of atomic motion has been widely accepted because it results in lower working and free Rs. I've seen it knock 3 to 5 percent off, and while that certainly means that the model fits the data better, I'm not sure that the quality of the hydrogen bond distances, van der Waals distances, or maps are any better. The latter details are what I really look for in a model. On the other hand, there has been good evidence through the years that there is useful information in the data beyond an I/sigI of 2 or an Rmeas> 100% but getting people to use this data has been a hard slog. The reason for this reluctance is that the R values of the resulting models are higher. Of course they are higher! That does not mean the models are of poorer quality, only that data with lower signal/noise has been used that was discarded in the models you used to develop your "gut feeling" for the meaning of R. When you change your criteria for selecting data you have to discard your old notions about the acceptable values of empirical quality measures. You either have to normalize your measure, as Phil Jeffrey recommends, by ensuring that you calculate your R's with the same reflections, or by making objective measures of map quality. Dale Tronrud P.S. It is entirely possible that refining a model to a very optimistic resolution cutoff and calculating the map to a lower resolution might be better than throwing out the data altogether. On 5/1/2012 10:34 AM, Kendall Nettles wrote: I have seen dramatic improvements in maps and behavior during refinement following use of the UCLA anisotropy server in two different cases. For one of them the Rfree went from 33% to 28%. I don't think it would have been publishable otherwise. Kendall On May 1, 2012, at 11:10 AM, Bryan Lepore wrote: On Mon, Apr 30, 2012 at 4:22 AM, Phil Evans<pre(a)mrc-lmb.cam.ac.uk<mailto:[email protected]>> wrote: Are anisotropic cutoff desirable? is there a peer-reviewed publication - perhaps from Acta Crystallographica - which describes precisely why scaling or refinement programs are inadequate to ameliorate the problem of anisotropy, and argues why the method applied in Strong, et. al. 2006 satisfies this need? -Bryan _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb

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