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Twinning Refinement
by Joseph Brock 26 Mar '10

26 Mar '10

Dear Phenix Users, I'm currently using version: Version: 1.4 Release tag: 3 cctbx tag: 2008_12_07_1353 Platform: mac-intel-osx osx-10.5.6 I am working on characterising substrate/inhibitor interactions of a protein, the structure has been solved many times so its unit cell dimensions, contents (1 molecule per ASU) and space group (P3121) are well characterized. However, I have recently encountered several crystals that appear exhibit merohedral twinning, and am having trouble converging the R-factors to reasonable levels. The details of one such data set is described below: Diffraction to ~1.9A was recorded using a high and low resolution pass of 180º each. When processed with HKL2000 separately or together, Scalepack wanted to reject huge amounts of my data (~20%). I was forced to increase the error scale factor to 2 and adjust my error model to the very high R-factors of the low-resolution shells. The summary table from my final round of scalepack is: Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 4.09 7648.3 1498.7 182.7 1.030 0.376 0.424 4.09 3.25 3885.1 568.9 64.1 1.410 0.416 0.480 3.25 2.84 1609.7 225.0 26.1 1.227 0.451 0.517 2.84 2.58 807.9 97.1 25.3 1.958 0.439 0.561 2.58 2.39 496.6 59.7 22.0 1.060 0.257 0.268 2.39 2.25 335.5 53.0 18.1 0.717 0.275 0.238 2.25 2.14 221.7 19.0 17.4 2.389 0.354 0.298 2.14 2.05 143.1 19.5 17.5 1.537 0.459 0.351 2.05 1.97 87.0 20.2 17.1 1.079 0.663 0.480 1.97 1.90 54.6 20.3 16.7 1.073 0.990 0.696 All reflections 1588.3 269.2 41.9 1.350 0.400 0.444 In addition, many systematic absences appear to be present: Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 5 177.7 56.7 3.1 0 0 7 255.5 70.6 3.6 0 0 8 264.8 73.2 3.6 0 0 10 595.0 164.2 3.6 0 0 11 2343.5 1289.9 1.8 0 0 13 321.3 125.4 2.6 0 0 14 66.5 21.8 3.1 0 0 16 238.0 76.3 3.1 0 0 17 394.3 109.1 3.6 0 0 19 13.0 7.1 1.8 0 0 20 650.1 179.5 3.6 0 0 22 199.6 55.6 3.6 0 0 23 953.9 372.0 2.6 0 0 25 192.2 62.2 3.1 0 0 26 1038.7 405.0 2.6 0 0 28 96.3 28.9 3.3 0 0 29 2303.0 897.0 2.6 0 0 31 12.0 11.5 1.0 0 0 32 1622.4 516.5 3.1 0 0 34 11.5 13.8 0.8 0 0 35 22.8 14.8 1.5 0 0 37 27.9 18.0 1.5 0 0 38 154.3 45.6 3.4 0 0 40 55.8 27.8 2.0 0 0 41 35.5 23.7 1.5 0 0 43 -25.3 34.4 -0.7 0 0 44 67.7 32.1 2.1 The data has an overall completeness of 99.7%. When I run the data trough xtriage, it tells me: Statistics depending on twin laws ----------------------------------------------------------------- | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | ----------------------------------------------------------------- | -h,-k,l | M | 0.307 | 0.301 | 0.256 | 0.022 | ----------------------------------------------------------------- Patterson analyses - Largest peak height : 5.288 (corresponding p value : 0.86552) The largest off-origin peak in the Patterson function is 5.29% of the height of the origin peak. No significant pseudotranslation is detected. The results of the L-test indicate that the intensity statistics are significantly different than is expected from good to reasonable, untwinned data. As there are twin laws possible given the crystal symmetry, twinning could be the reason for the departure of the intensity statistics from normality. It might be worthwhile carrying out refinement with a twin specific target function. I also estimated the twin fraction with the following server: http://nihserver.mbi.ucla.edu/Twinning/ And found it to about 20% (using data from 6-3A). After phasing the data with rigid body refinement in Refmac with intensity based twin refinement (brings both R-factors to ~36%), I have tried doing a twin refinement with Phenix: %phenix.refine model.pdb data.mtz twin_law="-h,-k,l" strategy=rigid_body+individual_sites+group_adp The R_factors consistently diverge to the following values: Final R-work = 0.3392, R-free = 0.4360 Simulated annealing/TLS/ordered solvent/messing with wxc/wxu weights does not seem to improve the situation. I have also tried this with and without prior detwinning with the CCP4 program "Detwin", with similar results. Reindexing to -h,-k,l gives similar R-factors after rigid body refinement but after refinement with Phenix, things get even worse: Final R-work = 0.3919, R-free = 0.5059 I was surprised to see that the fit to the map using both indexing conventions are comparable even though this should effectively reverse the hand of the structure. I assume this is because twin refinement is basically doing the job of "partial reindexing" on those parts of the lattice which are twinned? I have been working on this for about a week now and can't for the life of me find a way forward. If anyone can offer any suggestions It would greatly be appreciated. Im slowly beginning to lose my sanity.... BTW I don't think my problems stem from misindexing, although the beam was wondering all over the place during this block of data collection, I have previously solved another structure at the same d and xbeam ybeam values without any trouble. Sorry about the v.long post. Thought it would be better to give too much information rather than not enough =) Many thanks for all your suggestions in advance and for everything I have learned from this community in the past. Cheers, Joe PhD student. _________________________________________________________________ Get personal with Windows. Download a free gift for your PC. http://experience.windows.com

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project defaults ncs.params not read or overridden/overwritten by GUI?
by hari jayaram 25 Mar '10

25 Mar '10

Hi I am quite sure I am doing something wrong , but the refinement seems to always re-determine the NCS definitions despite it being set in the ncs.params file in the project defaults directory. Here is what I did: 1. I set the NCS groups definition in the GUI to go from residues 35:445 , 2. I check that using the view pick . Everything looks fine 3. I click the Green update and exit button 4. I look at the ncs.params file in the project defaults directory and it indeed reflects the changes . 5. I start the refinement from the GUI and the ncs section seems to have been reset to 26:450 . I even tried adding the ncs.params file to the Input files section, but to no avail. The ncs seems to always get re-determined and I end up getting an excessive distance stoppage of refinement. I even made sure I dont have the Find NCS restraints automatically button checked. Any idea why I cannot get the gui run refinement to remember my ncs definitions. Thanks in advance Hari

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FreeR label?
by J. Fleming 25 Mar '10

25 Mar '10

Hello all, I used AutoSol to generate a model from my Se-Met crystal. I then used this output model, overall_best.pdb, for AutoBuild1 in which I extended the data to include a set of native data. At this point I took the exptl_fobs_phases_freeR_flags.mtz output and used it with a different model that I believe to be accurate to carry out AutoBuild2. The problem I'm having is that I can't run Refmac/Phenix refinements on the output from AutoBuild2: overall_best.pdb and overall_best_denmod_map_coeffs.mtz. Both pieces of software say the FreeR labels are missing although in the AutoBuild2 output PDB there is a FreeR test set count of ~2,000. When I list all labels in Refmac I see: F FP P PHIM W FOMM Using these labels doesn't work for refinement. I used default settings for AutoSol/AutoBuild except for the number of Se-Met sites and of course the f' and f'' etc. I'm assuming the problem is with the MTZ but I'm not sure where to troubleshoot. Does anyone have any thoughts? Thanks in advance, Jon

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OS X PowerPC builds
by Ben Eisenbraun 24 Mar '10

24 Mar '10

Hi PHENIXians, I'm updating our nightly build, and I noticed that there are no current builds for OS X PowerPC. Are the PowerPC builds going to be discontinued? I searched the mailing list, but I didn't see an announcement. Thanks. -ben -- | Ben Eisenbraun | Software Sysadmin | | Structural Biology Grid | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |

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Bulk solvent scaling & unstructured protein
by Mads Beich-Frandsen 24 Mar '10

24 Mar '10

Hi all, I have a P1-crystal and data (quite anisotropic to 2.85Å, 90% complete) of a partly unstructured protein, of which some 35% of the sequence is intrinsically unstructured. The structure has been solved by molecular replacement, and it has been refined to Rw/Rf 20/26 with Phenix. As expected, not much of the predicted unstructured region show clear density. The oligomeric protein packs in layers facing oneanother, leaving the unstructured parts 'packing' towards eachother, in what appears as huge solvent-channels. My question is now: Is there an approach to optimizing bulk solvent scaling in Phenix, keeping with the thought, that 30-35% of my protein sequence should be present where Phenix assigns bulk solvent. Thank you! -And any other tricks and tips will much appreciated! Rgds., Mads ------------------------------ Mads Beich-Frandsen Department of Structural and Computational Biology Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria E-mail: mads.beich-frandsen(a)univie.ac.at Phone: +43-1-4277-52225 Fax: +43-1-4277-9522 Web: http://www.mfpl.ac.at/index.php?cid=58

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where to find phenix.phaser 2.2.3 documentation on keywords
by Bjørn Panyella Pedersen 24 Mar '10

24 Mar '10

Dear list I am struggling to find the documentation for Phaser 2.2.3 (as part of phenix.phaser v1.6.289), and more specifically the list of allowed keywords and what their significance is. In my hands the PACK keyword is no longer allowed in v2.2.3 (works fine in v2.1.4) while two alternatives: SELECT CUTOFF DISTANCE and QUICK are suggested instead. However I cannot find any details on what these do. Ultimately I want to allow packing clashes beyond the default of 10. A google-search only give me hits on the Phaser homepage with documentation for v.2.1 (http://www-structmed.cimr.cam.ac.uk/phaser/documentation/index.html), and the phenix.phaser documentation (http://www.phenix-online.org/documentation/phaser.htm) does not include a keyword-list. Hope somebody can help with this simple problem. Best Bjørn -- Bjørn Panyella Pedersen, PhD Postdoc PUMPKIN, Centre for Membrane Pumps in Cells and Disease University of Aarhus, Dept. Molecular Biology Gustav Wieds Vej 10C DK-8000 Aarhus C, Denmark Phone: +45 89425261 E-mail: bjopp(a)bioxray.au.dk http://www.bioxray.au.dk

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question about autosol
by crystallogrphy 22 Mar '10

22 Mar '10

Hi, When I run phenix.autosol with this scripts (phenix.autosol data=output-anomalous.sca input_phase_file=AutoMR_run_10_/MR.1.mtz input_phase_labels="F PHIC FOM" f_prime=-8.13 f_double_prime=5.61 kp-zntb.seq atom_type=Zn), the job is terminated by the error message. Does anyone know what I should do? I am using Phenix-1.5 version. Floating-point error (Python and libc call stacks above) This crash may be due to a problem in any imported Python module, including modules which are not part of the cctbx project. To disable the traps leading to this message, define these environment variables (e.g. assign the value 1): BOOST_ADAPTBX_FPE_DEFAULT BOOST_ADAPTBX_SIGNALS_DEFAULT This will NOT solve the problem, just mask it, but may allow you to proceed in case it is not critical.

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help with geometry target weight
by Brown, Breann 18 Mar '10

18 Mar '10

I am currently refining a structure at 2.4A resolution. I would like to increase the geometry target weight during refinement, however when I decrease wxc_scale (to 0.25 or 0.1) there is no change in the bond deviations or R factors compared to refinement with the default wxc_scale. I noticed in the refinement logfile under XYZ refinement wc=0. This does not change when I change the wc value in the command line (wc=2.0 for example) as suggested in a previous email. I also see no change when I use the optimize_wxc=true command. Examples of the command line input and output log files are below. I am currently using version 1.6-289 and would appreciate any help in resolving this issue as it seems the geometry is not being properly weighted during refinement. Thanks, Breann Breann L. Brown Department of Molecular Pharmacology, Physiology and Biotechnology Brown University Box GE-3 Providence, RI 02912 >phenix.refine data.mtz model.pdb ligand.cif twin_law strategy=tls+individual_adp tls.params main.ncs=true wxc_scale=0.1 wc=2.0 ========================== Target weights: x-ray data ========================= |-----------------------------------------------------------------------------| | XYZ refinement: T = Eexperimental * wxc * wxc_scale + Echem * wc | | wxc = 1.000000 wxc_scale = 0.100 wc = 0.000 | | angle between x-ray and geometry gradient vectors: None (deg) | | | | ADP refinement: T = Eexperimental * wxu * wxu_scale + Eadp * wu | | wxu = 11.057053 wxu_scale = 2.160 wu = 1.000 | | angle between Xray and ADP gradient vectors: 143.484 (deg) | |-----------------------------------------------------------------------------| ------------------------------------------------------------------------ stage angl bond chir dihe plan repu geom_target 0 : 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 1_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 1_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 2_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 2_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 3_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 3_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 4_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 4_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 5_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 5_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 6_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 6_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 7_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 7_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 8_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 8_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 9_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 9_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 10_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 10_adp: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 10_bss: 2.493 0.027 0.239 23.648 0.008 4.106 3.6797e-01 ------------------------------------------------------------------------ Maximal deviations: stage angl bond chir dihe plan repu |grad| 0 : 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 1_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 1_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 2_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 2_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 3_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 3_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 4_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 4_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 5_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 5_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 6_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 6_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 7_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 7_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 8_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 8_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 9_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 9_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 10_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 10_adp: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 10_bss: 29.287 0.287 2.517 83.486 0.050 1.724 3.9155e-01 -----Original Message----- From: phenixbb-bounces(a)phenix-online.org [mailto:[email protected]] On Behalf Of Pavel Afonine Sent: Tuesday, February 23, 2010 1:40 PM To: PHENIX user mailing list Subject: Re: [phenixbb] weights optimization, add hydrogens Hi Ursula, > I am refining a 2.0 A structure in Phenix using mostly the graphical > interface. The maps are very good and the R factors are low > (0.208/0.173). But the geometry is not so great with bond deviations > of 0.208 and Angle deviations of 1.85. What is the best thing to do > to try to improve the geometry. Should I try to optimize the weights? the R-factors seem good indeed. Yes, Although the angle deviations seem ok to me, I'm concerned about the bond deviations... Yes, you can try two things: - increase the restraints strength by increasing the corresponding weight: from the command line: "wc=VALUE", where VALUE is a number somewhat larger than 1.0; from the GUI: Refinement settings -> All parameters -> Refinement target weights... -> Stereochemistry weight scale - Run automatic weight optimization (note, this may take a while to run): from the command line: use keyword: "optimize_wxc=true"; from the GUI: Refinement settings -> All parameters -> Refinement target weights... -> Optimize X-ray/stereochemistry weight Alternatively, you can send me your PDB and data file and I will tell you what exactly is not right and how to fix it. Pavel.

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Re: [phenixbb] phenix.simple_ncs_from_pdb output... OMAT?
by Thomas C. Terwilliger 17 Mar '10

17 Mar '10

Hi Francis, No, I don't think it is the same as OMAT format.Here is the format from phenix.simple_ncs_from_pdb (identical to the format from resolve): (Mapping molecule j onto molecule 1) (As input) Operator # 1 New X-prime= 1.0000 X + 0.0000 Y + 0.0000 Z + 0.0000 New Y-prime= 0.0000 X + 1.0000 Y + 0.0000 Z + 0.0000 New Z-prime= 0.0000 X + 0.0000 Y + 1.0000 Z + 0.0000 Approximate center_of_mass of this object (from center_of_mass of object 1 and NC symmetry) is -28.39 18.81 -16.36 Operator # 2 New X-prime= -1.0000 X + -0.0035 Y + -0.0040 Z + -4.1335 New Y-prime= 0.0043 X + -0.1070 Y + -0.9943 Z +-21.6192 New Z-prime= 0.0031 X + -0.9943 Y + 0.1070 Z +-19.2559 Approximate center_of_mass of this object (from center_of_mass of object 1 and NC symmetry) is 24.44 -7.12 -39.79 would be represnted as: rota_matrix 1.0000 0.0000 0.0000 rota_matrix 0.0000 1.0000 0.0000 rota_matrix 0.0000 0.0000 1.0000 tran_orth 0.0000 0.0000 0.0000 center_orth -28.3915 18.8125 -16.3621 rota_matrix -1.0000 -0.0035 -0.0040 rota_matrix 0.0043 -0.1070 -0.9943 rota_matrix 0.0031 -0.9943 0.1070 tran_orth -4.1335 -21.6192 -19.2559 center_orth 24.4419 -7.1171 -39.7930 This is in contrast to OMAT: O/RAVE Format OMAT r11 r21 r31 (note that the rotation matrix is r12 r22 r32 transposed with respect to CCP4 r13 r23 r33 or conventional matrix format) t1 t2 t3 where x' = r11 x + r12 y + r13 z + t1 y' = r21 x + r22 y + r23 z + t2 z' = r31 x + r32 y + r33 z + t3 I hope that helps! -Tom T >> Is the output rota_matrix (x3), and tran_orth(x1) in OMAT format? >> >> Thanks >> >> F >> >> --------------------------------------------- >> Francis Reyes M.Sc. >> 215 UCB >> University of Colorado at Boulder >> >> gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D >> >> 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D >> >> >> >> _______________________________________________ >> phenixbb mailing list >> phenixbb(a)phenix-online.org >> http://phenix-online.org/mailman/listinfo/phenixbb >>

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phenix.simple_ncs_from_pdb output... OMAT?
by Francis E Reyes 17 Mar '10

17 Mar '10

Is the output rota_matrix (x3), and tran_orth(x1) in OMAT format? Thanks F --------------------------------------------- Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D

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