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refinement of partly occupied water molecules?
by krimmer＠staff.uni-marburg.de 30 May '13

30 May '13

Dear all, I have a question concerning the refinement of partly occupied water molecules: in some of my macromolecular crystal structures with resolutions between 1.1 - 1.4 Å, mainly in the second solvation shell round positive Fo-Fc electron density blobs are detectable which show after assignment of a water molecule to these blob and subsequent refinement with Phenix refine a good-looking calculated 2Fo-Fc electron density (round shape, the 2Fo-Fc map appears at a contour level of σ = 1.0 - 1.4), but there also occurs a small negative Fo-Fc electron density detectable inside the 2Fo-Fc density blob. The negative Fo-Fc electron density disappears if the occupancy of the water molecule is refined by Phenix or manually set lower than 100% occupancy. Therefore, I think these positions are partly occupied by water molecules, but I am not sure how I should handle it/how it is generally handled. I would be thankful for any advice and/or literature about this topic. My second question is concerning a density blob in perfect coordination distance to an indolyl group (perfectly located in the middle of the five-ring, with 3.4 Å distance to every of the five carbon atoms of the five-ring). What‘s the best way to find out which ion possibly occupies this position? I tried Na+ and Ca2+, but after refinement big negative Fo-Fc blobs appear in both cases (maybe the ion at this position is only partly occupied?). There is no anomalous signal detectable at this position (data collection wavelength was 0.91841 nm). After the refinement of a water molecule at this position, a positive Fo-Fc election density is still there. Thank you for your help! Stefan

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R-merge
by Mengbin Chen 29 May '13

29 May '13

Dear Phenix Users, I have R-merge values equal 0.000 from highest resolution shell (which corresponds to values larger than 1.000) in a data set processed by HKL2000, with redundancies higher than 10 (I know this may help in increasing R-merge). I am not sure if this is going to be a problem of Table 1 for the reviewers. Could you kindly drop me your suggestions? Thank you in advance, Mengbin Chen -- Mengbin Chen Department of Chemistry University of Pennsylvania

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phenix.refine using anomalous concepts for neutron data?
by Derek Logan 29 May '13

29 May '13

Hi, I'm doing a joint X-ray neutron refinement in Phenix and have run into some behaviour that puzzles me. I had not been paying any attention to the use or non-use of anomalous signal in the neutron dataset, as This is what I get if I let phenix.refine itself decide whether or not to use anomalous data: ================================= Neutron data ================================ F-obs: neutron.mtz:FO,SIGFO,DANO,SIGDANO,ISYM Miller array info: neutron.mtz:FO,SIGFO,DANO,SIGDANO,ISYM Observation type: xray.reconstructed_amplitude Type of data: double, size=16672 Type of sigmas: double, size=16672 Number of Miller indices: 16672 Anomalous flag: True Unit cell: (removed) Space group: P 21 21 21 (No. 19) Systematic absences: 0 Centric reflections: 1258 Resolution range: 29.22 1.89826 Completeness in resolution range: 0.785008 Completeness with d_max=infinity: 0.78486 Bijvoet pairs: 6832 Lone Bijvoet mates: 1750 Anomalous signal: 0.0876 Number of F-obs in resolution range: 16672 Number of F-obs<0 (these reflections will be rejected): 0 Number of F-obs=0 (these reflections will be used in refinement): 0 Refinement resolution range: d_max = 29.2200 d_min = 1.8983 and this is what I get if I forcibly switch off use of anomalous data in the phenix.refine GUI: ================================= Neutron data ================================ F-obs: neutron.mtz:FO,SIGFO,DANO,SIGDANO,ISYM Miller array info: neutron.mtz:FO,SIGFO,DANO,SIGDANO,ISYM Observation type: xray.reconstructed_amplitude Type of data: double, size=16672 Type of sigmas: double, size=16672 Number of Miller indices: 16672 Anomalous flag: True Unit cell: (removed) Space group: P 21 21 21 (No. 19) Systematic absences: 0 Centric reflections: 1258 Resolution range: 29.22 1.89826 Completeness in resolution range: 0.785008 Completeness with d_max=infinity: 0.78486 Bijvoet pairs: 6832 Lone Bijvoet mates: 1750 Anomalous signal: 0.0876 force_anomalous_flag_to_be_equal_to=False Reducing data to non-anomalous array. R-linear = sum(abs(data - mean(data))) / sum(abs(data)) R-square = sum((data - mean(data))**2) / sum(data**2) In these sums single measurements are excluded. Redundancy Mean Mean Min Max Mean R-linear R-square unused: - 29.2234 bin 1: 29.2234 - 4.0863 1 2 1.699 0.0240 0.0011 bin 2: 4.0863 - 3.2448 1 2 1.793 0.0302 0.0022 bin 3: 3.2448 - 2.8350 1 2 1.800 0.0417 0.0041 bin 4: 2.8350 - 2.5760 1 2 1.764 0.0486 0.0054 bin 5: 2.5760 - 2.3914 1 2 1.745 0.0505 0.0051 bin 6: 2.3914 - 2.2505 1 2 1.717 0.0513 0.0051 bin 7: 2.2505 - 2.1378 1 2 1.658 0.0533 0.0056 bin 8: 2.1378 - 2.0448 1 2 1.614 0.0611 0.0074 bin 9: 2.0448 - 1.9661 1 2 1.575 0.0634 0.0075 bin 10: 1.9661 - 1.8983 1 2 1.485 0.0736 0.0097 unused: 1.8983 - Fobs statistics after all cutoffs applied: Miller array info: None Observation type: xray.amplitude Type of data: double, size=9840 Type of sigmas: double, size=9840 Number of Miller indices: 9840 Anomalous flag: False Unit cell: (removed) Space group: P 21 21 21 (No. 19) Systematic absences: 0 Centric reflections: 1258 Resolution range: 29.22 1.89826 Completeness in resolution range: 0.855801 Completeness with d_max=infinity: 0.855503 What's going on here? If I let phenix.refine decide automatically, it looks like it's reading in F+ and F- separately and regarding them as independent in the refinement. If I force "no anomalous" it merges them. However I can't work out whether in either case it has actually read FOBS instead of the Friedel mates. According to the SCALA manual the I+ and I- columns will always be written out even if the keyword ANOMALOUS OFF is used, so this is a potential pitfall for many. In addition, phenix.refine seems to identify the neutron data first as xray.reconstructed_amplitude then as xray.amplitude even though the data are explicitly defined as neutron data in the GUI. I can send more information if required! Thanks Derek ________________________________________________________________________ Derek Logan tel: +46 46 222 1443 Associate Professor mob: +46 76 8585 707 Dept. of Biochemistry and Structural Biology www.cmps.lu.se<http://www.cmps.lu.se> Centre for Molecular Protein Science www.maxlab.lu.se/node/307<http://www.maxlab.lu.se/node/307> Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com<http://www.saromics.com>

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Structure Comparison questions
by Heather Condurso 29 May '13

29 May '13

Dear all, I have a few related structures that I would like to compare to make figures for publication but I have a few issues. The biggest issue is that my structures are large (12- 422aa chains) and any attempt at simple alignment in pymol results in a crash. I would like to first compare entire structures, but also compare the differences between related chains in the same structure. I want to look at the small differences in the active site density. What is the best way to do this? Is there a simple way to do this or do I need to make separate pdb files for each chain? Can I use the same mtz or should I rename copies? I will try a few things but any advice would be greatly appreciated. Heather condurso(a)bc.edu

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R: problem refining RNA structure at 4Å resolution
by fulvio.saccoccia＠uniroma1.it 29 May '13

29 May '13

Any signs of twinning? Check also the correctness of your space group: probably you could index In a lower symmetry Laue group with a slight increased distorsion index. Fulvio ----Messaggio Originale---- Da: Benoît Masquida Inviato: 29/05/2013, 14:53 A: PHENIX user mailing list Oggetto: [phenixbb] problem refining RNA structure at 4Å resolution Dear phenix users, I solved using MR a low resolution RNA structure taking data up to 4.3Å and a model of a structure with slight differences. However, when I try to refine, Rwork decreases quickly (start 47, final 32 but Rfree remains high (start 48, final 44). The data are pretty good up to 4.0 with I/sigma above 1. The map doesn't point clearly to wrong regions of the model. What can I try to improve the maps and build the correct model? Thanks in advance. Benoit _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org http://phenix-online.org/mailman/listinfo/phenixbb

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problem refining RNA structure at 4Å resolution
by Benoît Masquida 29 May '13

29 May '13

Dear phenix users, I solved using MR a low resolution RNA structure taking data up to 4.3Å and a model of a structure with slight differences. However, when I try to refine, Rwork decreases quickly (start 47, final 32 but Rfree remains high (start 48, final 44). The data are pretty good up to 4.0 with I/sigma above 1. The map doesn't point clearly to wrong regions of the model. What can I try to improve the maps and build the correct model? Thanks in advance. Benoit

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Re: [phenixbb] paired refinement in phenix gui
by Kay Diederichs 28 May '13

28 May '13

On Mon, May 27, 2013 at 15:08:26 -0700, Nat Echols wrote: >> >It would be nice if you could confirm if my settings are ok, or if there are >> >still some settings in the background which I overlooked, because we see >> >small changes in R and Rfree. > Changes relative to what? It is not expected that feeding the output > PDB file back to phenix.refine (or another program) will exactly > reproduce the R-factors from the previous round of refinement, because > the precision of the atomic parameters is limited by PDB format. > (Using mmCIF instead would be more reliable, but we don't think a > change in R-factor of 0.0004 is worth worrying about anyway.) > >> >I added the model refined at higher resolution (e.g. 1.5A) and the mtz file >> >cut at CC (e.g.1.5A) in the ?Input data? tab, and set High resolution cutoff >> >in ?X-ray data and experimental phases? box to (e.g. 1.8A) >> >In the refinement setting strategy tab I uncheck everything and set Number >> >of cycles = 1. >> >In ?other options? I uncheck everything too. > This sounds correct, but it's much easier to simply run the validation > GUI, because the underlying code (specifically phenix.model_vs_data) > is designed to calculate identical R-factors to what phenix.refine > shows you. From what I've seen, recent phenix.model_vs_data works correctly and reproduces the statistics from phenix.refine within the numerical accuracy that one can expect. > >> >Alternatively adding a file to the input data with > I don't recommend this - using parameter files as input is fine for > large repetitive parameter blocks such as custom geometry restraints, > selections from the TLSMD server, or nucleic acid base pairs, but > anything that attempts to override the controls in the main GUI may > not work. > >> >2. For whatever reason I would like to do that (molprobity) - can I (and how >> >can I do this) add the command line commands also in the gui? > Could you please clarify? > >> >3. In december 2012 there was a discussion about weak data and refinement. I >> >would like to know if there are new data available showing that ?Whether the >> >benefit they describe is considered cosmetic or non-trivial,.....? > Maybe Kay could comment on this... There is not a single answer that fits all situations. When done correctly (e.g. keeping Rfree flags, keeping refinement strategy), in those cases that I looked at, using weak reflections (cutoff at <I/sigma>~0.5 to 1, cc1/2~0.15-0.25) improved the model's Rfree at lower resolution (paired refinement!) by a few percent, compared to cutting the data at I/sigma>2. Just try it and see if the improvement is worth it, according to your own judgement! > >> >4. Additionally could one short comment on what phenix is doing with strong >> >(weak) reflections marked as aliens in XDS. > Assuming that XDS doesn't actually remove these, phenix.refine may or > may not discard them at the outlier filtering stage (which can be > disabled). I don't know how the different methods for flagging > suspect resolutions compare; Kay and/or Pavel would know more about > this. The "Alien" list in CORRECT.LP was only (originally) meant to alert the user to ice reflections, zingers, and cosmic rays (not sure if the latter play a role, but the former certainly do!). If the "Alien" list just shows reflections at very high resolution, in low-<intensity> resolution shells, then this is due to the fact that Wilson statistics does not hold in these shells, and therefore the algorithm for deciding about "Aliens" is no longer valid. If this happens, you should (as identified in analysis together with James Holton) prevent CORRECT from marking "Aliens" as outliers by changing REJECT_ALIEN from 20 (the default) to 100 or so. Otherwise the mean intensity in that resolution shell will drop (due to rejecting the strongest reflections), and that is bad for applying the Wilson&French algorithm. The XDSCONV in the latest XDS package (identifying itself as March-30-2013) has two fixes for dealing with weak data (again, based on an exchange with James Holton). Unfortunately, a small bug (affecting very few datasets) was identified by Graeme Winter (thanks!) in the XDS program of that package, so be prepared to download the XDS package again in a few days. hope that helps, Kay > >> >According to the supplementary of P. Andrew Karplus, Kay Diederichs ?Linking >> >Crystallographic Model and Data Quality? >> >I would like to do >> >anisotropic refinement, with additional options >> >?ordered_solvent.new_solvent=anisotropic >> >adp.individual.anisotropic="not element H" >> >fix_rotamers=True?. > If your resolution really is 1.5?, I wouldn't recommend making waters > anisotropic. There is some difference of opinion whether anisotropic > refinement of protein atoms is acceptable; it's probably good to try > both ways. (It would be interesting to know whether including the > weak high-resolution data allows you to parameterize the model more > aggressively.) > > -Nat > -- Kay Diederichs http://strucbio.biologie.uni-konstanz.de email: Kay.Diederichs(a)uni-konstanz.de Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature "smime.p7s".

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PDB deposition
by Kay Diederichs 27 May '13

27 May '13

Dear Phenix developers, the following questions came up today, during an attempt to answer the questions that the PDB asks at deposition time: a) For Bulk solvent modeling, what should be given as value for K sol and B sol ? My understanding is that the new solvent model does no longer result in these values; the table is Total model structure factor: F_model = k_total * (F_calc + k_mask * F_mask) k_total = k_isotropic * k_anisotropic Resolution Compl Nwork Nfree R_work <Fobs> <Fmodel> kiso kani kmask 37.014-13.525 85.34 95 4 7.2146 38.755 318.867 0.937 1.000 0.300 13.240-10.663 91.82 91 10 7.1453 40.328 328.027 0.901 1.000 0.309 10.640-8.570 93.27 202 6 7.4011 38.069 318.901 0.905 1.002 0.310 8.548-6.875 95.70 380 21 7.2679 31.166 258.647 0.901 1.000 0.309 6.871-5.523 95.40 731 36 7.1964 29.485 241.669 0.908 1.001 0.302 5.520-4.439 90.49 1347 61 7.3445 39.504 329.514 0.934 1.001 0.281 4.438-3.568 92.52 2610 150 7.3166 41.793 347.063 0.961 1.000 0.280 3.567-2.867 94.10 5191 231 7.2983 33.593 278.704 0.965 1.000 0.024 2.867-2.304 92.97 9835 476 7.3098 23.922 198.755 0.984 1.000 0.000 2.304-1.852 92.81 18869 947 7.3097 18.746 155.751 1.053 1.000 0.000 1.852-1.488 90.33 35341 1797 7.3288 10.914 90.931 1.035 0.999 0.000 1.488-1.196 86.49 64979 3561 7.2975 6.848 56.810 1.028 0.998 0.000 1.196-1.081 53.28 29672 1567 7.1776 5.351 43.737 0.986 1.000 0.000 Approximation of k_total with k_overall*exp(-b_overall*s**2/4) k_overall=1.0149 b_overall=-0.0072 So what can we do here? b) there is the question about Overall Anisotropic values B11; B22; B33; B12; B13; B23.... but the logfile offers overall anisotropic scale matrix: V0: -0.0214,0.0415,-0.0249,0.0006,-0.0515,-0.0161 V1: -0.0055,-0.0022,0.0080,-0.0001,-0.0020,0.0011 So which of these should be given? I've tried to locate the information about these two questions in the documentation, but it seems I missed it! thanks, Kay

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command line molprobity tools
by Engin Özkan 27 May '13

27 May '13

Hi everybody, I want to create a comprehensive, human-readable log file by running validation using molprobity tools within phenix command-line. I do not have structure factors/maps. Apparently, the GUI runs validation through phenix.model_vs_data (according to the .eff file), but in command-line, I cannot get phenix.model_vs_data to run without an mtz/hkl. I guess I could provide it a fake data file, and then set the comprehensive flag to "True"? BTW, the log file produced by the GUI is not comprehensive at all. I could not find a related mmtbx or cctbx function either. Another mentioned tool, phenix.validate, does not even exist in my installation (dev 1361). Thanks, Engin -- Engin Özkan Post-doctoral Scholar Laboratory of K. Christopher Garcia Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 w ph: (650)-498-7111 cell: (650)-862-8563

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Fwd: [ccp4bb] "Formageddon" is upon us... Important news from wwPDB!
by Paul Adams 24 May '13

24 May '13

Begin forwarded message: > From: Gerard DVD Kleywegt <gerard(a)XRAY.BMC.UU.SE> > Subject: [ccp4bb] "Formageddon" is upon us... Important news from wwPDB! > Date: May 24, 2013 11:20:51 AM PDT > To: CCP4BB(a)JISCMAIL.AC.UK > Reply-To: info(a)wwpdb.org > > Dear colleagues, > > I would like to draw your attention to a notification from the wwPDB partners about "Deposition and Release of PDB Entries Containing Large Structures" - see: > > http://www.wwpdb.org/news/news_2013.html#22-May-2013 > > There are major changes afoot in the way large structures are handled in the PDB, as well as in the deposition and annotation procedures and software used by the wwPDB sites. This is of immediate relevance for depositors and users of large structures, but also for software developers and anyone who routinely processes the entire PDB archive or its weekly releases (bioinformatics resources, etc.). From 2014, it will affect essentially everyone who deposits, uses or processes PDB entries. > > If you have any questions about the new deposition system or the procedures for handling large structures or any of the other changes, please contact: info(a)wwpdb.org > > Please pass on this information to anyone likely to be affected by the upcoming changes. Thanks! > > > On behalf of the Worldwide Protein Data Bank, > > --Gerard Kleywegt > > --- > Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK > gerard(a)ebi.ac.uk ..................... pdbe.org > Secretary: Pauline Haslam pdbe_admin(a)ebi.ac.uk -- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Division Deputy for Biosciences, Advanced Light Source, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Laboratory Research Manager, ENIGMA Science Focus Area Building 64, Room 248 Building 80, Room 247 Building 978, Room 4126 Tel: 1-510-486-4225, Fax: 1-510-486-5909 http://cci.lbl.gov/paul Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. Executive Assistant: Louise Benvenue [ LBenvenue(a)lbl.gov ][ 1-510-495-2506 ] --

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