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Fo-Fo map
by Xiang Li 21 Jul '13

21 Jul '13

Hi everyone, I am working on two datasets of the similar crystal design. In one of the datasets, there is an extra guest protein (might be considered as a ligand) docked in one unit cell. The two crystals have very similar unit cell size and same symmetry. I want to calculate the Fo-Fo map to see whether I could figure out where the protein is. But I got error says the symmetry of the two datasets are not the same. I have never used the "isomorphous difference map" in GUI before. Can anyone help me to figure out where I did wrong please? I have attached the mtz file that I want to compare. Btw, if the protein is not very rigidly linked with the crystal framework (~2 free residues linkage), am I supposed to see the density of the protein? Or are there any better ways for me to identify the protein? Thanks for your kind help! Sincerely, Xiang

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ADP refinement of cysteinesulfonic acid
by Phillip Steindel 16 Jul '13

16 Jul '13

Hi all- I have a 1.5 Angstrom structure with two cysteines that appear to have oxidized to cysteinesulfonic acid (OCS). I replaced the cysteines in coot, then ran refine with individual and anisotropic ADPs, but the ADPs don't appear to be refining for those residues. The hydrogens are all at 24,and the non-hydrogens are all at 20. I'm running 1.8.2-1309. Thanks, Phillip Steindel Theobald Lab Brandeis University

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cis peptide question
by Edward A. Berry 16 Jul '13

16 Jul '13

I have a cis-peptide in my structure. Phenix recognizes it and .geo file shows this is restrained to 0*: dihedral pdb=" CA ALA A 401 " pdb=" C ALA A 401 " pdb=" N SER A 402 " pdb=" CA SER A 402 " ideal model delta harmonic sigma weight residual 0.00 -1.58 1.58 0 5.00e+00 4.00e-02 1.00e-01 However in statistics printed with the output pdb, I have at least one dihedral that is flagged as off by about 180*: REMARK 3 DEVIATIONS FROM IDEAL VALUES. REMARK 3 RMSD MAX COUNT REMARK 3 DIHEDRAL : 12.902 168.987 3277 ^^^^^^^ Could this be my cis peptide, or do I have a really bad dihedral somewhere else?

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How do I turn off sidechain rotamer fitting?
by Owen Pornillos 15 Jul '13

15 Jul '13

I'm using one of the nightlies - dev 1427. It's not doing very well in combination with NCS. Or what is the most recent nightly that does not have this feature? Many thanks, Owen

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Kicked difference maps
by Ho Leung Ng 12 Jul '13

12 Jul '13

Hi, I am finding that features in kicked difference maps (mFo-DFc) have very different scaling compared to the standard difference maps. For example, a blob that appears in the standard map at 3 sigma might not appear in the kicked map until 5.5 sigma. Is this correct functioning? In contrast, features in the kicked and standard 2mFo-DFc maps are comparable in scale. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry hng(a)hawaii.edu

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problem with alternate conformations
by George Phillips 11 Jul '13

11 Jul '13

I am trying to adapt my old CNS scripts to to multiple conformer refinements in Phenix, i.e. up to 20 copies of each entire chain. I seem to be OK with up to three copies of the whole chain, but when I try four or more I seem to be in an infinite loop in the 'reduce' phase of a refinement. 3 copies runs in less than hour, even with torsion simulated annealing, with four even overnight the program just runs 'reduce for hours and hours'. This happens on the standard release and development version 1422. Is there some way to turn off hydrogens as a work around? Test files available on request. : ) Thanks for any help! George George N. Phillips, Jr. Ralph and Dorothy Looney Professor Department of Biochemistry and Cell Biology Rice University, MS140 6100 Main Street Houston, Texas 77005-1892 Office: +1 713.348.6951 http://www.bioc.rice.edu/

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2014 Gordon Research Conference and Research Seminars on Diffraction Methods in Structural Biology
by Anastassis Perrakis 11 Jul '13

11 Jul '13

Dear all, In a bit more than a year from now, we will be running the Gordon Research Conference on Diffraction Methods in Structural Biology, this time preceded for the first time by a two-day Gordon Research Seminar, exclusively for young scientists and a few mentors. http://www.grc.org/programs.aspx?year=2014&program=diffrac http://www.grc.org/programs.aspx?year=2014&program=grs_diff We are in the process of inviting the right people to make the schedule as exciting and inviting as possible, hoping we will be able to attract a diverse, enthusiastic, and motivated crowd! At this stage, we would like to ask you for ideas for 1. Specific subjects that are emerging and you would like to see covered, to make it worth your time to join us! 2. Specific people that would be excellent speakers, that would make you attend the Research Conference! 3. Specific people that would be great mentors, that would make you attend the Research Seminars! Many thanks from the team (but please send all replied to me!) Tassos Perrakis (GRC chair), Eddie Snell (GRC vice chair), Jeff Headd (GRS chair) and Maike Bublitz (GRS assoc. chair)

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parallel phenix.phaser on SGE cluster
by L. Costenaro (IBB) 11 Jul '13

11 Jul '13

Hello, I am trying to run phaser in a SGE cluster (qsub) using multiple proc (either phenix.phaser from the command line or phaser-MR from the GUI), but the jobs do not parallelize. When I run the phaser-MR locally (same executable) it does parallelize (multiple python threads). Any help , advice would be welcome. Best regards, Lionel

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Location of Gaussian factors for ED calculation
by Schubert, Carsten [JRDUS] 10 Jul '13

10 Jul '13

Hi, Were in the phenix package can I find the Gaussian factors (scattering table) for calculating the isotropic ED distribution at a given radius of r. I just need that for an illustration. Even better of you could already point me to some plots. Nothing I found online so far was suitable. Thanks Carsten

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: Post-doctoral position in structural biology (Paris, France)
by Benas Philippe 10 Jul '13

10 Jul '13

POST-DOCTORAL POSITION IN STRUCTURAL BIOLOGY (PARIS, FRANCE) . A postdoctoral position is available in the group of Dr. Carine Tisne at the Laboratory of Biological Crystallography and NMR of the University Paris Descartes, jointly with the group of Dr. Ciaran Condon at the Laboratory of Microbial Gene Expression (CNRS UPR9073) of the Institut de Biologie Physico-Chimique. We are studying the structure, function and folding of RNA and their complexes using NMR spectroscopy and X-ray crystallography, together with complementary biophysical methods. The postdoctoral research project focusses on the processing of the 5 and 3 ends of 5S rRNA in Gram-positive bacteria using solution NMR and X-ray crystallography as the main techniques. An integral part of our projects is the use and development of state-of-the-art preparative, biochemical and NMR techniques. . Interested candidates should hold a Ph.D. in Biochemistry, Biophysics or Structural Biology. Expertise in NMR spectroscopy and/or X-ray crystallography is mandatory. The successful candidate should be highly motivated, creative and capable of independent research in a two-team environment (10 minute walk between labs). The position is for a two-year term contract (one year in each team). We offer an exciting and enthusiastic work environment together with a stimulating location at the center of Paris (close to Jardin du Luxembourg). Facilities for structural biology include 600 MHz Bruker spectrometer equipped with cryoprobe. A new 700 MHz spectrometer will be installed in 2014. We also have access to high-field NMR spectrometers through the French TGIR and regular access to synchroton beamtime at either the ESRF or SOLEIL as Beam Time Allocation Group member. The ideal start date for the position would be October 2013. . Applications and informal queries about the labs and research projects should be directed by email to Carine Tisne (carine.tisne(a)parisdescartes.fr) or Ciaran Condon (condon(a)ibpc.fr[1]). Applications must include a letter of motivation summarizing current and future research interests, a CV, list of publications and letters of recommendation from at least two references. . ------------------------- PHILIPPE BENAS, PH.D. X-ray diffraction and computing facilities manager Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS E-mails: philippe.benas(a)parisdescartes.fr, philippe_benas(a)yahoo.fr URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18 ------------------------- "We do know nothing ! But tomorrow is another day ! ;-) " Liens: ------ [1] mailto:[email protected]

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give-up-quick Phaser in Rosetta
by Morten Grøftehauge 10 Jul '13

10 Jul '13

Dear Phenixbb, I was wondering if there was any way of getting Rosetta to give up easily and move on to another search model? Say I have 20 or more equally awful search models. I think there's a possibility that one of them will knock it out of the park (i.e. TFZ > 8). But even if I have Phaser set to fast search I will end up with an exhaustive search because the search models are so bad. fast_search_mode= True Run phaser with selection_criteria_rot_value and then if no obvious solution, repeat with cutoff lowered by search_down_percent search_down_percent= 25 Used if fast_search_mode=True. Run phaser with selection_criteria_rot_value and then if no obvious solution, repeat with cutoff lowered by search_down_percent If I set search_down_percent to 75 does this mean that Phaser will give up easily and move on to the next model? Is this a terrible strategy CPU-time-wise? I am of course aware that search_down_percent is much more likely to give me the right solution for a given model but in this case there are just so many possible search models. Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University

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inconsistent Rwork/Rfree in outputs
by Michael Thompson 09 Jul '13

09 Jul '13

Hello All, I am getting an output from phenix.refine that is somewhat confusing to me. I have a log file that seems to be reporting inconsistent values for the final Rwork and Rfree. The last line of my log file says: Final R-work = 0.1815, R-free = 0.1912 However, when I check the pdb file from phenix.refine, I see: REMARK Final: r_work = 0.1799 r_free = 0.1952 bonds = 0.012 angles = 1.315 In the log file, I get the following table, which seems to agree with what is in the pdb file. R-factors, x-ray target values and norm of gradient of x-ray target stage r-work r-free xray_target_w xray_target_t 0 : 0.1971 0.1916 1.408780e-01 1.965297e-01 1_bss: 0.1971 0.1916 1.508155e-01 2.122150e-01 1_ohs: 0.1971 0.1916 1.447084e-01 2.026856e-01 1_xyz: 0.1928 0.2001 1.348543e-01 2.162596e-01 1_adp: 0.1929 0.2134 1.006068e-01 1.652532e-01 1_occ: 0.1928 0.2127 9.708801e-02 1.642701e-01 2_bss: 0.1784 0.1978 1.001979e-01 1.536656e-01 2_ohs: 0.1786 0.1982 1.146147e-01 1.876601e-01 2_xyz: 0.1777 0.1959 1.187570e-01 1.899659e-01 2_adp: 0.1952 0.2001 1.109064e-01 1.588435e-01 2_occ: 0.1952 0.1995 1.059382e-01 1.580417e-01 3_bss: 0.1831 0.1888 1.066004e-01 1.508888e-01 3_ohs: 0.1833 0.1890 1.217008e-01 1.855605e-01 3_xyz: 0.1824 0.1897 1.256200e-01 1.855714e-01 3_adp: 0.1876 0.2039 1.027854e-01 1.533022e-01 3_occ: 0.1877 0.2042 9.739524e-02 1.525993e-01 4_bss: 0.1770 0.1931 9.830779e-02 1.515598e-01 4_ohs: 0.1772 0.1933 1.132096e-01 1.824877e-01 4_xyz: 0.1775 0.1933 1.179076e-01 1.860704e-01 4_adp: 0.1911 0.2005 1.074584e-01 1.546149e-01 4_occ: 0.1909 0.1997 1.023003e-01 1.530269e-01 5_bss: 0.1805 0.1895 1.034638e-01 1.506407e-01 5_ohs: 0.1807 0.1895 1.185810e-01 1.828381e-01 5_xyz: 0.1803 0.1899 1.244219e-01 1.801964e-01 5_adp: 0.1893 0.2019 1.072974e-01 1.503622e-01 5_occ: 0.1893 0.2023 1.016701e-01 1.492730e-01 6_bss: 0.1792 0.1915 1.021896e-01 1.490341e-01 6_ohs: 0.1794 0.1915 1.174694e-01 1.798680e-01 6_xyz: 0.1804 0.1926 1.224254e-01 1.830383e-01 6_adp: 0.1952 0.2015 1.118187e-01 1.549205e-01 6_occ: 0.1950 0.2017 1.063759e-01 1.538692e-01 7_bss: 0.1842 0.1922 1.076805e-01 1.529198e-01 7_ohs: 0.1845 0.1918 1.227818e-01 1.857086e-01 7_xyz: 0.1846 0.1917 1.292627e-01 1.856928e-01 7_adp: 0.1885 0.2042 1.071531e-01 1.532154e-01 7_occ: 0.1885 0.2044 1.012764e-01 1.524448e-01 7_bss: 0.1797 0.1952 1.353649e-01 1.556126e-01 7_ohs: 0.1799 0.1952 1.353649e-01 1.556126e-01 I'm wondering why this last line of the log file isn't consistent with the other values that are being reported for R/Rfree? Which numbers should I believe? Thanks, Mike -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles miket(a)chem.ucla.edu

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GUI admin issues
by Yarrow Madrona 08 Jul '13

08 Jul '13

Hello, I have a couple of GUI related questions 1. We recently moved all of our data from one hard drive to another. However, we do not know how to change the directory for all of our projects. The directory is similar but the hard drive is now called tp3home instead of tp2home. We found a .phenix directory that has a file with a list of projects and directories. We updated the directories but this did not make a difference in the GUI. 2. Is there a quick way to specify which ligand or amino acid to remove in the GUI with doing simmulated annealing or better to do manually? Thanks. -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697

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Re: [phenixbb] omit map for protein-DNA complex
by Wei Shi 08 Jul '13

08 Jul '13

Hi guys, With the help of Tom and Nat, I fixed the errors in my pdb file and could generate the simulated annealing omit map for the protein-DNA complex structure! But when I opened resolve_composite_map.mtz and *_ed.pdb in Coot, I could only see one monomer of the protein in Coot. It should be two dimers of the protein plus two pieces of DNA. And when I opened the *_ed.pdb in pymol, it showed the right thing: two dimers of the protein plus two pieces of DNA. It seems that something in *_ed.pdb file is not recognized by Coot. I don't know whether any of you had this before and would have any suggestions for me. When I opened *_ed. pdb with text file and found out that the word "break" is used instead of "ter" between different chains, so I replaced all "break" with "ter", but this didn't help.... Thank you so much! Best, Wei On Wed, Jul 3, 2013 at 10:36 AM, Wei Shi <wei.shi118(a)gmail.com> wrote: > Hi Nat and Tom, > Thank you so much for your help! I don't know why those C7 atoms are > there.... I just added some nucleotides manually using coot to fit the > density. I am going to delete those C7 atoms in DA and see whether that > would help. Thank you guys so much! > > Best, > Wei > > On Tue, Jul 2, 2013 at 5:22 PM, Nathaniel Echols <nechols(a)lbl.gov> wrote: > >> Okay, this makes sense. Wei, are you trying to model methylated DNA, >> or are those C7 atoms there by accident? >> >> -Nat >> >> On Tue, Jul 2, 2013 at 12:11 PM, Terwilliger, Thomas C >> <terwilliger(a)lanl.gov> wrote: >> > Hi Nat, >> > I think it was the input file. Here is a little piece of it: >> > >> > ATOM 3157 P DA E 8 -56.682 44.723-118.292 1.00 37.45 >> P >> > ATOM 3158 C5' DA E 8 -54.313 44.743-117.204 1.00 40.32 >> C >> > ATOM 3159 O5' DA E 8 -55.729 44.807-117.015 1.00 38.38 >> O >> > ATOM 3160 C4' DA E 8 -53.551 44.795-115.886 1.00 35.58 >> C >> > ATOM 3161 O4' DA E 8 -53.433 46.172-115.429 1.00 32.68 >> O >> > ATOM 3162 C3' DA E 8 -54.164 44.027-114.709 1.00 30.30 >> C >> > ATOM 3163 O3' DA E 8 -53.117 43.597-113.848 1.00 30.92 >> O >> > ATOM 3164 C2' DA E 8 -54.997 45.106-114.029 1.00 18.46 >> C >> > ATOM 3165 C1' DA E 8 -53.974 46.233-114.120 1.00 19.87 >> C >> > ATOM 3166 N1 DA E 8 -52.933 50.956-112.506 1.00 4.27 >> N >> > ATOM 3167 C2 DA E 8 -52.179 49.849-112.633 1.00 5.31 >> C >> > ATOM 3168 N3 DA E 8 -52.515 48.630-113.069 1.00 3.59 >> N >> > ATOM 3169 C4 DA E 8 -53.816 48.619-113.401 1.00 5.26 >> C >> > ATOM 3170 C5 DA E 8 -54.722 49.662-113.344 1.00 5.31 >> C >> > ATOM 3171 C6 DA E 8 -54.237 50.894-112.863 1.00 4.84 >> C >> > ATOM 3172 N6 DA E 8 -55.010 51.981-112.750 1.00 5.38 >> N >> > ATOM 3173 C7 DA E 8 -56.909 56.086-110.227 1.00 2.18 >> C >> > ATOM 3174 N7 DA E 8 -55.973 49.258-113.789 1.00 6.11 >> N >> > ATOM 3175 C8 DA E 8 -55.803 47.992-114.102 1.00 7.27 >> C >> > ATOM 3176 N9 DA E 8 -54.525 47.556-113.888 1.00 8.54 >> N >> > ATOM 3177 OP1 DA E 8 -56.215 43.588-119.122 1.00 44.46 >> O >> > ATOM 3178 OP2 DA E 8 -58.094 44.785-117.853 1.00 32.00 >> O >> > >> > Notice the C7 that is not attached to anything and is not normally part >> of a DA nucleotide. >> > -Tom T >> > >> > On Jul 2, 2013, at 1:07 PM, Nathaniel Echols wrote: >> > >> >> On Tue, Jul 2, 2013 at 9:03 AM, Wei Shi <wei.shi118(a)gmail.com> wrote: >> >>> Below is the error message I got when I ran a plain refinement of the >> same >> >>> PDB file and data file. I also ran phenix.ready-set of the PDB file >> using >> >>> default setting (add hydrogen to model if absent, et al) and generate >> a >> >>> updated PDB file and ran refine again, but still got the same error >> message >> >>> as below: >> >> >> >> This sounds like a bug. Could you please email us off-list with the >> >> PDB file and any existing restraints? (We can fake the data for >> >> testing purposes.) >> >> >> >> -Nat >> > >> > >

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Re: [phenixbb] problem refining Fe-S cluster
by Edward A. Berry 05 Jul '13

05 Jul '13

Pavel Afonine wrote: > Hi Ed, > > it looks like occupancies and B-factors need to be refined and refined > until convergence. Also, if you apply any distance restraints for > clusters (restrain Fe-S distances) make sure these restraints are > correct. I've seen many cases like this and typically refining > occupancy/b-factors and making sure restraints are correct solves the > problem. I've never seen fixing specific parameters help and I can't > understand logic for doing this. > OK. I'm not giving any restraints for the cluster, it must be in the monomers library. I can get SF4.cif from CCP4 and set the weights for bonds/angles very low to let the density rule. But now I see the problem wasn't there a few rounds back, and the difference seems to be that I was using 3 macro_cyles instead of 1. Now I repeat exactly the same refinement both ways, seems to be it. So maybe an artifact of not refining to convergence. Odd though, because it was OK in the starting model, and I'm not asking for the B's to be reset initially to 20 or something. Problem seems to be solved by using the default 3 cycles, but in case anyone is interested, B-factors for Fe are pretty much unchanged after refining 3 cycles B-factors for the Fe's but not the S's, decrease by about 5.0 on refining 1 cycle. Overall and protein B-ave increase on refining 1 cycle Looking at the step by step changes, the first cycle out of 3 is not like the single cycle. This is dev-1420 version of phenix. R-factors refining 3 cycles REMARK R-factors, x-ray target values and norm of gradient of x-ray target REMARK stage r-work r-free xray_target_w xray_target_t REMARK 0 : 0.3205 0.3532 3.857969e+00 3.920754e+00 REMARK 1_bss: 0.2433 0.2823 3.683390e+00 3.779722e+00 REMARK 1_weight: 0.2433 0.2823 3.683390e+00 3.779722e+00 REMARK 1_xyzrec: 0.2457 0.2825 3.689060e+00 3.780966e+00 REMARK 1_adp: 0.2463 0.2823 3.690454e+00 3.780932e+00 REMARK 1_occ: 0.2463 0.2823 3.690455e+00 3.780936e+00 REMARK 2_bss: 0.2463 0.2823 3.690087e+00 3.780536e+00 REMARK 2_weight: 0.2463 0.2823 3.690087e+00 3.780536e+00 REMARK 2_xyzrec: 0.2439 0.2821 3.684528e+00 3.778960e+00 REMARK 2_adp: 0.2434 0.2823 3.683429e+00 3.779315e+00 REMARK 2_occ: 0.2434 0.2823 3.683429e+00 3.779316e+00 REMARK 3_bss: 0.2433 0.2825 3.683615e+00 3.779760e+00 REMARK 3_weight: 0.2433 0.2825 3.683615e+00 3.779760e+00 REMARK 3_xyzrec: 0.2455 0.2825 3.688717e+00 3.780899e+00 REMARK 3_adp: 0.2461 0.2824 3.689934e+00 3.780715e+00 REMARK 3_occ: 0.2461 0.2824 3.689934e+00 3.780714e+00 REMARK end: 0.2457 0.2825 3.689963e+00 3.781089e+00 refining 1 cycle REMARK stage r-work r-free xray_target_w xray_target_t REMARK 0 : 0.3205 0.3532 3.857969e+00 3.920754e+00 REMARK 1_bss: 0.2222 0.2930 3.668217e+00 3.804573e+00 REMARK 1_weight: 0.2222 0.2930 3.668217e+00 3.804573e+00 REMARK 1_xyzrec: 0.2284 0.2941 3.681902e+00 3.808504e+00 REMARK 1_adp: 0.2594 0.3133 3.748285e+00 3.841014e+00 REMARK 1_occ: 0.2594 0.3133 3.748284e+00 3.841012e+00 REMARK end: 0.2692 0.2912 3.759155e+00 3.806820e+00 REMARK ------------------------------------------------------------------------ R B-factors refining 3 cycles REMARK |-----overall-----|---macromolecule----|------solvent-------| REMARK stage b_max b_min b_ave b_max b_min b_ave b_max b_min b_ave REMARK 0 : 90.34 16.23 43.10 90.34 16.23 42.91 80.33 27.29 46.57 REMARK 1_bss: 90.40 16.29 43.15 90.40 16.29 42.97 80.39 27.35 46.63 REMARK 1_weight: 90.40 16.29 43.15 90.40 16.29 42.97 80.39 27.35 46.63 REMARK 1_xyzrec: 90.40 16.29 43.15 90.40 16.29 42.97 80.39 27.35 46.63 REMARK 1_adp: 89.27 16.38 42.96 89.27 16.38 42.79 80.39 26.98 46.24 REMARK 1_occ: 89.27 16.38 42.96 89.27 16.38 42.79 80.39 26.98 46.24 REMARK 2_bss: 89.37 16.47 43.06 89.37 16.47 42.88 80.49 27.08 46.34 REMARK 2_weight: 89.37 16.47 43.06 89.37 16.47 42.88 80.49 27.08 46.34 REMARK 2_xyzrec: 89.37 16.47 43.06 89.37 16.47 42.88 80.49 27.08 46.34 REMARK 2_adp: 89.46 16.19 42.92 89.46 16.19 42.74 80.48 26.86 46.22 REMARK 2_occ: 89.46 16.19 42.92 89.46 16.19 42.74 80.48 26.86 46.22 REMARK 3_bss: 89.63 16.36 43.09 89.63 16.36 42.92 80.66 27.03 46.39 REMARK 3_weight: 89.63 16.36 43.09 89.63 16.36 42.92 80.66 27.03 46.39 REMARK 3_xyzrec: 89.63 16.36 43.09 89.63 16.36 42.92 80.66 27.03 46.39 REMARK 3_adp: 89.22 16.53 42.92 89.22 16.53 42.75 80.45 26.77 46.08 REMARK 3_occ: 89.22 16.53 42.92 89.22 16.53 42.75 80.45 26.77 46.08 REMARK end: 89.22 16.53 42.92 89.22 16.53 42.75 80.45 26.77 46.08 refining 1 cycle: REMARK |-----overall-----|---macromolecule----|------solvent-------| REMARK stage b_max b_min b_ave b_max b_min b_ave b_max b_min b_ave REMARK 0 : 90.34 16.23 43.10 90.34 16.23 42.91 80.33 27.29 46.57 REMARK 1_bss: 90.40 16.29 43.15 90.40 16.29 42.97 80.39 27.35 46.63 REMARK 1_weight: 90.40 16.29 43.15 90.40 16.29 42.97 80.39 27.35 46.63 REMARK 1_xyzrec: 90.40 16.29 43.15 90.40 16.29 42.97 80.39 27.35 46.63 REMARK 1_adp: 85.40 23.38 49.20 85.40 23.38 49.17 81.25 33.88 49.84 REMARK 1_occ: 85.40 23.38 49.20 85.40 23.38 49.17 81.25 33.88 49.84 REMARK end: 85.40 23.38 49.20 85.40 23.38 49.17 81.25 33.88 49.84 REMARK ------------------------------------------------------------------------ R starting model oswego 236% grep SF4 sqr1402e_002.pdb ATOM 8568 FE4 SF4 B1003 15.699 21.265 101.440 1.00 31.53 Fe ATOM 8569 FE1 SF4 B1003 15.302 20.444 104.268 1.00 28.32 Fe ATOM 8570 FE3 SF4 B1003 15.072 18.385 101.975 1.00 30.57 Fe ATOM 8571 FE2 SF4 B1003 17.702 19.420 102.964 1.00 32.57 Fe ATOM 8572 S2 SF4 B1003 14.066 20.264 102.463 1.00 39.50 S ATOM 8573 S3 SF4 B1003 16.963 21.440 103.248 1.00 28.46 S ATOM 8574 S1 SF4 B1003 16.723 19.393 100.994 1.00 31.08 S ATOM 8575 S4 SF4 B1003 16.026 18.425 103.963 1.00 29.56 S refining 3 cycles oswego 235% grep SF4 sqr1402e_006.pdb ATOM 8568 FE4 SF4 B1003 15.708 21.268 101.432 1.00 31.37 Fe ATOM 8569 FE1 SF4 B1003 15.283 20.449 104.291 1.00 28.14 Fe ATOM 8570 FE3 SF4 B1003 15.062 18.373 101.986 1.00 30.15 Fe ATOM 8571 FE2 SF4 B1003 17.720 19.419 102.961 1.00 32.34 Fe ATOM 8572 S2 SF4 B1003 14.080 20.264 102.462 1.00 38.64 S ATOM 8573 S3 SF4 B1003 16.943 21.422 103.257 1.00 28.96 S ATOM 8574 S1 SF4 B1003 16.712 19.383 101.004 1.00 31.27 S ATOM 8575 S4 SF4 B1003 16.036 18.446 103.959 1.00 29.32 S refining 1 cycle oswego 234% grep SF4 sqr1402e_005.pdb ATOM 8568 FE4 SF4 B1003 15.763 21.282 101.383 1.00 26.59 Fe ATOM 8569 FE1 SF4 B1003 15.274 20.434 104.305 1.00 23.38 Fe ATOM 8570 FE3 SF4 B1003 15.026 18.351 102.006 1.00 25.63 Fe ATOM 8571 FE2 SF4 B1003 17.779 19.437 102.902 1.00 27.63 Fe ATOM 8572 S2 SF4 B1003 14.132 20.305 102.437 1.00 34.56 S ATOM 8573 S3 SF4 B1003 16.929 21.408 103.255 1.00 28.42 S ATOM 8574 S1 SF4 B1003 16.681 19.335 101.002 1.00 29.49 S ATOM 8575 S4 SF4 B1003 16.105 18.462 103.923 1.00 28.25 S

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problem refining Fe-S cluster
by Edward A. Berry 05 Jul '13

05 Jul '13

I have a problem refining a structure with iron-sulfur clusters. The irons refine to low B-factors (~25 in this case, while wilson B and B-average are around 45 and the Sg's ligating the irons are ~30). The irons have negative difference density and there is positive density in the center of the cluster (not on the S as it appears; the S is actually above the plane of the irons and the positive density below). (see attached map) It's possible that the cluster is lost in some percent of the molecules, and I could refine occupancy to get rid of the negative density, but I suspect this is likely to be because the B-factors are too low. I will try manually setting B for the Fe's and refining without ADP step, but does anyone have a better suggestion? This was refined for one macrocycle with strategy = *individual_sites individual_sites_real_space rigid_body \ *individual_adp group_adp tls *occupancies group_anomalous thanks, eab

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divergence of R and Rfree at the last step of refinement
by Edward A. Berry 03 Jul '13

03 Jul '13

I'm refining a structure with phenix-dev-1414 version, and R-free decreases until the last step where R-work drops sharply and R-free increases. Am I doing something wrong? R-factors, x-ray target values and norm of gradient of x-ray target stage r-work r-free xray_target_w xray_target_t 0 : 0.3068 0.3347 4.111964e+00 4.184576e+00 1_bss: 0.2296 0.2735 3.919523e+00 4.036648e+00 1_weight: 0.2296 0.2735 3.919523e+00 4.036648e+00 1_xyzrec: 0.2377 0.2725 3.938818e+00 4.036514e+00 1_adp: 0.2311 0.2707 3.918953e+00 4.031556e+00 1_occ: 0.2311 0.2707 3.918944e+00 4.031541e+00 end: 0.2187 0.2795 3.907777e+00 4.050961e+00 ////////////////////////////////////////////////////////////////// Looking in the log file, it seems that this happens at the stage of correcting the solvent mask: ============================= updating all scales ============================= start: r_work=0.3152 r_free=0.3389 (reset all scales to undefined) bulk-solvent and scaling: r_work=0.2324 r_free=0.2703 remove outliers: r_work=0.2324 r_free=0.2703 *-> correct solvent mask: r_work=0.2187 r_free=0.2795 <--*** final: r_work=0.2187 r_free=0.2795 overall anisotropic scale matrix: V0: 2.5130,-0.3626,1.3424,-0.7174,-0.0625,-2.3276 V1: -0.0348,-0.0556,-0.0105,-0.0091,0.0085,0.0052

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Very low resolution data set
by Xiang Li 03 Jul '13

03 Jul '13

Hi everyone, I am working on a very low resolution data set (14 A). It is a designed DNA crystal and unit cell size & symmetry fit well with the assumption. When I do the refinement I found the Rfree is extreme high, ~0.49 while Rwork is about 0.4. But the model seems fits in well with the density map. I have collected different datasets, and it gives about the same MR result for different sets. I do not have any experience to work with low resolution data. Here I have a few questions: 1 What is the expected Rfree and Rwork for such low resolution data? I looked up PDB bank, and there is a 11.5A crystal with Rfree=~0.4. But not sure whether there is a kind of value for Rfree to consider the refinement works or not. 2 There might be overfit problem during the refinement. Currently what I did is to optimize the weight with PHENIX gui, add restraints between basepairs and NCS restraint. I tried to manually adjust the weight, but seems the optimized weight gives the best result. Are there other things I should try? 3 How to confirm that the result is real? There will be larger than usual error for such low resolution data, and what the supposed range should be? Thanks a lot for your suggestions and help! Sincerely, Xiang

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omit map for protein-DNA complex
by Wei Shi 02 Jul '13

02 Jul '13

Hi guys, I've been trying to generate the omit map for a protein-DNA complex structure using Phenix. I don't know why it takes forever to run and didn't seem like to finish at all.... The resolution of the dataset is 3.2A. Omit map type: simulated annealing. Omit region: composite. And, for the corresponding apo-protein structure, the simulated annealing omit map was generated within 2 or 3 hours on the same computer cluster. Let me know whether you have any idea about what might go wrong and whether there is anything I could do.... Thank you so much! Best, Wei

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Bias in Rfree
by "Weiergräber, Oliver H." 02 Jul '13

02 Jul '13

Hello, while refining a structure with NCS, I was re-considering this paper from Michael Chapman's group: Fabiola F, Korostelev A, Chapman MS. Bias in cross-validated free R factors: mitigation of the effects of non-crystallographic symmetry. Acta Crystallogr D Biol Crystallogr. 2006 Mar;62(Pt 3):227-38. In essence, they suggest to exclude from the working set reflections that are (1) related to a test reflection by NCS (2) adjacent to a test reflection and its NCS mates in reciprocal space They have implemented this strategy in a CNS module (R-free2005), but the software is no longer actively supported. Does anybody know if something similar has ever been implemented in other software packages such as phenix? Or am I missing any significant downsides of the approach - apart from a reduction of the working set? Best regards, Oliver ================================================ PD Dr. Oliver H. Weiergräber Institute of Complex Systems ICS-6: Structural Biochemistry Tel.: +49 2461 61-2028 Fax: +49 2461 61-9540 ================================================ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Forschungszentrum Juelich GmbH 52425 Juelich Sitz der Gesellschaft: Juelich Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498 Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher Geschaeftsfuehrung: Prof. Dr. Achim Bachem (Vorsitzender), Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt, Prof. Dr. Sebastian M. Schmidt ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------

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Error while updating water
by Subhani Bandara 01 Jul '13

01 Jul '13

Dear all, I am refining a protein structure at 2.5 A. After running some refinements with water update option on, I manually added water to some positions and ran without water update option as it removes those water from the sites and show green density around. Then after some refinements like this, I tried to use water update option to add water to some newly appeared density. When I check the option to update water using dev 1418 version it stops with the following error. Also when not using water update option, refinement statistics got worse when I used 1418 compared to 1.8.2-1309 with refinements using same inputs and options. R work-0.1988 compared to 0.1920 with 1.8.2-1309 Rfree-0.2623 compared to 0.2490 with 1.8.2-1309 Traceback: File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/cctbx_project/libtbx/thread_utils.py", line 236, in run return_value = self._target(self._args, self._kwargs, self._c) File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/cctbx_project/libtbx/runtime_utils.py", line 94, in __call__ result = self.target() File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/cctbx_project/libtbx/runtime_utils.py", line 73, in __call__ result = self.run() File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/runtime.py", line 69, in run refine_object = self._run(args, callback) File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/runtime.py", line 85, in _run call_back_handler=callback) File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/command_line.py", line 161, in run call_back_handler=call_back_handler) File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/driver.py", line 1630, in run ncs_manager = self.ncs_manager).result File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/strategies.py", line 155, in __init__ neutron_refinement2 = self.neutron_refinement2) File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/task_table.py", line 26, in __init__ self.__call__() File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/task_table.py", line 36, in __call__ self.__getattribute__(method_name)() File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/task_table.py", line 106, in run_sol mc = self.macro_cycle) File "/Applications/PHENIX-dev-1418/Contents/phenix-dev-1418/phenix/phenix/refinement/task_table.py", line 79, in _need_to_run else: raise RuntimeError("Not a valid mode: %s"%mode) Thanks in advance for any help Subhani

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automatic weight optimization
by Michael Thompson 30 Jun '13

30 Jun '13

Hello Phenix Folks, I have a few questions about the automatic weight optimization in phenix.refine. I have read the manual, but I feel like maybe I'm missing something. I apologize in advance if these are naive questions, but here goes. I ran phenix.refine with a .def file, in which I set the following parameters: target_weights { optimize_xyz_weight = True optimize_adp_weight = True wxu_scale = 2.72 } My goal was to optimize both xyz and adp weights. In the log file, I see something like this: ===================== xyz refinement (lbfgs minimization) ===================== R-FACTORS RMSD CLASH RAMA ROTA CBET WEIGHT TARGETS work free delta bonds angl data restr 21.06 20.38 -0.67 0.033 2.0 6.1 0.0 6.6 0 none 0.154 0.1685 19.79 21.26 1.47 0.012 1.3 7.6 0.0 5.8 0 2.494 0.131 0.0499 Legend: - first line corresponds to starting state (before refinement) - R-factors reported in percent - delta is Rfree-Rwork in percent - CLASH is all-atom Molprobity clashscore - ROTA is percent of side-chain rotamer outliers - RAMA is percent of Ramachandran plot outliers - CBET is number of Cbeta deviations - WEIGHT is relative weight between X-ray (or neutron) target and restraints - TARGETS: the values of X-ray (or neutron) and restraints target functions ================================ ADP refinement =============================== ----------Individual ADP refinement---------- R-FACTORS <Bi-Bj> <B> WEIGHT TARGETS work free delta data restr 19.79 21.26 1.47 1.909 20.062 0.661 0.131 19.80 21.28 1.49 1.761 20.011 0.007 0.130 19.77 21.41 1.64 1.838 19.884 0.030 0.125 20.30 22.63 2.34 2.369 19.168 0.122 0.111 20.17 22.82 2.64 2.886 18.929 0.243 0.107 20.07 23.04 2.97 3.771 18.737 0.365 0.104 20.04 23.14 3.10 4.106 18.751 0.486 0.103 20.00 23.19 3.19 4.496 18.794 0.608 0.103 19.98 23.23 3.25 4.846 18.851 0.729 0.102 19.97 23.27 3.29 5.115 18.940 0.851 0.102 19.95 23.30 3.36 5.420 18.988 0.972 0.101 19.94 23.33 3.39 5.579 19.008 1.094 0.101 19.93 23.29 3.36 5.598 18.917 1.215 0.101 max suggested <Bi-Bj> for this run: 10.00 max allowed Rfree-Rwork gap: 5.0 range of equivalent Rfree: 0.0 Best ADP weight: 0.007 Accepted refinement result: 19.80 21.28 1.49 1.761 20.011 0.007 0.130 The output kind of makes me feel like the adp weight is being optimized, but the xyz weight might not be. There is a list of statistics for varying values of the adp weight, but there is no similar list in the output from the xyz refinement stages. So my questions are: Is the xyz weight being optimized? Did I miss something in the input that is required to turn on xyz weight optimization? If the weight is indeed being optimized, then I must be misinterpreting the log file. How can I check to make sure the xyz weight optimization is running? Thanks for the help!! Mike Thompson -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles miket(a)chem.ucla.edu

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Format problem of restraint file
by Xiang Li 30 Jun '13

30 Jun '13

Hi everyone, I generated the basepairing restraint file by command phenix.secondary_structure_restraints MR.1.pdb > ss.eff . Then I opened the ss.eff file with gedit and edit the basepairing information. However, after I finished editing and saved the file, the refine program can not read the file. The error is "This application does not support the file type for ss.eff (format:txt)". I have attached a copy of the eff file. Do I have a way to solve the problem? Thanks for your help! Sincerely, Xiang

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unwanted disulfids and geometry restraints
by Ursula Schulze-Gahmen 30 Jun '13

30 Jun '13

I have 2 questions: 1) I am using phenix.refine on the command line with a .eff file as input that was modified from previous GUI phenix runs. My structure has several Zn atoms and readyset creates a geometry_restraints file for the Zn interactions. I am not sure if phenix.refine gets these restraints when I run it from the command line. Do I need to read this file in some specific way? 2) The other question is: phenix refines several of the Cysteine neighbors in the Zn environment as disulfids, but they are not. How do I prevent this from happening? Ursula -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220

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Re: [phenixbb] what about heavy atom as model for MR
by zhaoy 29 Jun '13

29 Jun '13

Hi, It's my pleasure to recieved so much wonderful advice and I am trying according to the suggestions. I have used phenix.explore_metric_symmetry to see if there is any relationship between tetragonal and orthorhombic lattices. But the result suggests there is no relationship. In addition, I have some questions about the molecular replacement when density map entered as model. How to define clash between the model and How the value of RMS error of the density model impact the final MR result? Thank you! -- Yan Zhao, M.Phil. National Laboratory of Protein Sciences Institute of Biophysics Chinese Academy of Sciences 15 Datun Rd. Beijing, 100101 China Randy Read 写: Sorry, I'm coming a bit late to this thread, and most things I would have suggested have already been covered by Tom and Paul. Certainly there are examples in the literature of people using MR to place a heavy atom substructure against the anomalous differences, but as Tom pointed out there's the complication of making sure that you get the Se sites from one molecule and not from a combination of symmetry-related molecules, which probably won't have the same relative arrangement in the other crystal form. The one remaining thing I'd like to explore is whether you're sure that you've carried out the molecular replacement with density properly. This has become easier (with Tom's phenix.cut_out_density program), but it's still relatively complicated and error-prone. First, you have to be sure that you've cut out the density essentially for one molecule, and not a mixture of symmetry-related molecules. If the location and rough shape of the molecule isn't clear in your map, you may have to try things like cutting out spheres of density from different places in the unit cell and seeing which gives the clearest MR solution. Second, you have to make sure that you place the density in a reasonably large unit cell, 2.5-4 times the extent of the density that you've cut out, before using it as an MR model. Third, you have to choose an appropriate value for the effective RMS error of the density model. If your phases go to 4A, then a number like 1.5A to 2A might be appropriate. In our tests, the map can be pretty noisy but you can still get a very clear signal from the MR search. Of course, if the phase information is very weak, it still may not work even with an optimal envelope. Best wishes, Randy Read On 23 Jun 2013, at 15:56, "Terwilliger, Thomas C" <terwilliger(a)lanl.gov> wrote: You could try both the isomorphous and the anomalous differences. Each one has some useful information. You can even combine them but I'm guessing it won't help too much. Paul Adams' suggestion to check out related space groups is a very good one. Phenix.autosol only tries the opposite hand, so you have to try all the different screw axis possibilities that are not related by inversion. Another thing you could try is to repeat your MR with density from your map ( against the 2.6 A native) and get a long list of solutions. For each solution take the phases after MR and use them to calculate an anomalous difference Fourier for your semet P2221 data. Then notice if there are peaks in this map, and whether these peaks have the same relationship to the MR-placed density as you saw in your original map. If yes, you may have a solution. All the best, Tom T From: phenixbb-bounces(a)phenix-online.org [phenixbb-bounces(a)phenix-online.org] on behalf of zhaoy [zhaoy(a)moon.ibp.ac.cn] Sent: Sunday, June 23, 2013 3:32 PM To: PHENIX user mailing list Subject: Re: [phenixbb] what about heavy atom as model for MR Thank you very much. I will try as you said. The P2221 dataset's qulity are not very well. The points from diffraction image are not sharp. Besides, native dataset is 2.6A, setmet anomalous datasets is only 4A. So I have not obtained the solution with p2221datasets. There is only one molecular per AUS at p4322. So, from the initial map, I can confirm the heavy atom sites from the same molecular. Maybe I have a general idea about the method you said. But I still puzzled which F difference should I use, isomorphous or anomalous differences? Thanks! -- Yan Zhao, M.Phil. National Laboratory of Protein Sciences Institute of Biophysics Chinese Academy of Sciences 15 Datun Rd. Beijing, 100101 China Terwilliger, Thomas C 写: I'm not sure if there is a tool that will create the file you want for this. You could however just write a little script that reads in a .sca file and calculates F from I, subtract F+ - F- to get Dano, square that, write out Dano** 2 and sigma(Dano**2) as another .sca file. Then you could use this as input to molecular replacement with your sites from the P4322 dataset. There is an important caveat to this approach however: your sites from the P4322 have to be all part of the same molecule, and this might or might not happen automatically. If some sites are from one molecule and others from another molecule, then in the other crystal form their relationship won't be correct. If there is only one molecule in the au then this might or might not be a big problem. If there are more than one it is very likely to be a big problem. This would apply to the density search that you carried out as well. If your density is good enough to see where the molecule is, you might be able to figure out which of the symmetry-related sites to include in your MR search. Nevertheless as this is easy it seems like a fine thing to do. However I am wondering if it will work because your 2.6 A semet anomalous dataset did not yield a solution by itself, which is a little surprising... Also of course if your P4322 solution is not actually right or even just not close enough it would not work. -Tom T From: phenixbb-bounces(a)phenix-online.org [phenixbb-bounces(a)phenix-online.org] on behalf of 赵岩 [zhaoy(a)moon.ibp.ac.cn] Sent: Sunday, June 23, 2013 7:52 AM To: phenixbb(a)phenix-online.org Subject: [phenixbb] what about heavy atom as model for MR Hi everyone, The heavy atoms(Se-Met) were located at P4322 space group(4A). But the initial density map is so bad that I can not build the initial model. Meanwhile, I have another two datasets, native(2.6A) and se-met anomalous dataset with space group P2221. But these two datasets have no solution. I have tried the initial map from sp P4322 as a model to do molecular replacement. But no MR solution. So, I want to know how can I use the heavy atom as model to do MR with isomorphous or anomalous differences? Thanks! -- Yan Zhao, M.Phil. National Laboratory of Protein Sciences Institute of Biophysics Chinese Academy of Sciences 15 Datun Rd. Beijing, 100101 China _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org http://phenix-online.org/mailman/listinfo/phenixbb ------ 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 Road E-mail: rjr27(a)cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

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