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tls refinement
by sbiswas2＠ncsu.edu 11 May '09

11 May '09

Hi all, I started doing TLS refinement with one of my structures and got the following: Start R-work = 0.3817, R-free = 0.3923 (no bulk solvent and anisotropic scale) Final R-work = 0.3788, R-free = 0.3885 (no bulk solvent and anisotropic scale) Start R-work = 0.2094, R-free = 0.2381 Final R-work = 0.2058, R-free = 0.2358 my question now is why is the R value high without the anisotropic scale? Second question is: can I refine alternate conformations using the command line or is it already there in the default parameters? Thanks, Shya NC State University

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Phaser, Phenix and Em models
by Gino Cingolani 11 May '09

11 May '09

Hi Randy, what about implementing the use of EM-models (e.g. structure factors from EM model) in Phenix to carry out MR and/or MR-SAD? There's a growing number of macromolecular complexes that have been imaged at medium/low res by EM, which could be of great help to phase at low res or located anomalous scatterers in phased maps. And the EM database keeps growing! At this moment, it is certainly possible to use EM models in Phaser, but the Phenix pipeline could add much more to that. For instance, Phenix would allow for better phase extension of low res EM phases to higher res by ncs-averaging or multi-crystal averaging (in Resolve). Or Phenix could help combining low res EM phases with other phase sources such as SeMet sites, poorly occupied heavy atom sites, etc (MR-SAD). Or Phenix could rigid body refine (at low resolution) pseudo-atomic models obtained by filling in EM-models with dummy atoms. This would help defining averaging masks and so on. What do you think? Thanks in advance, Gino *********************************************************** Gino Cingolani, Ph.D. Assistant Professor Dept. of Biochemistry and Molecular Biology SUNY Upstate Medical University 750 E. Adams Street, Syracuse, NY, 13210 Tel. (315) 464 8744 Fax. (315) 464 8750 Email: cingolag(a)upstate.edu *********************************************************** "Nati non foste per viver come bruti, ma per seguir virtute e conoscenza" ("You were not born to live like brutes, but to follow virtue and knowledge") Dante, The Divine Comedy (Inferno, XXVI, vv. 119-120)

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Re: [phenixbb] Phaser, Phenix and Em models
by Gino Cingolani 11 May '09

11 May '09

Hi Randy, thanks for getting back to my email. Yes, I do think it would be very helpful to switch to using density instead of coordinates both for MR and MR-SAD. The interface between cryo-EM and X-ray is growing so fast that in many cases EM densities could be used for initial phasing by Fourier difference, or even ab initio, provided sufficient ncs is available. In our specific case we have at least two macromolecular complexes (1.1-2.2MDa) with >400SeMet, resolutions in the mid 3A with extreme unisomorphism and 10-15A EM densities. SAD/MAD phasing is unrealistic, MIR methods can't work when all crystals are inherently unisomorphic .... MR/MR-SAD could be an alternative. Thanks again! Gino Hi, As it happens, we've been working very hard over the last few months first to make all commands in Phaser available from Python (done) and second to make all options available from the new Phenix GUI (getting close). ("We" in this context mostly means Airlie at our end and Nat at the Berkeley end.) So it should become easy to switch to using density instead of coordinates for your models without switching to the command-line mode of running Phaser. We're also very interested in optimizing the way that we use density from EM, which is something we haven't spent enough time playing with yet. It's not yet possible to use density for a model in the MR-SAD mode--that's been on the wish list and shouldn't be too hard. Do you need that feature right now? I really like your other ideas, so we'll have to talk to Tom about adapting the AutoMR wizard so that it doesn't require a coordinate file. As you say, this will be a real growth area! All the best, Randy On 8 May 2009, at 15:39, Gino Cingolani wrote: > Hi Randy, > > what about implementing the use of EM-models (e.g. structure factors > from EM model) in Phenix to carry out MR and/or MR-SAD? > There's a growing number of macromolecular complexes that have been > imaged > at medium/low res by EM, which could be of great help to > phase at low res or located anomalous scatterers in phased maps. > And the EM database keeps growing! > > At this moment, it is certainly possible to use EM models in Phaser, > but the Phenix pipeline could add much more to that. For instance, > Phenix would allow for better phase extension of low res EM phases > to higher > res by ncs-averaging or multi-crystal averaging (in Resolve). > Or Phenix could help combining low res EM phases with other phase > sources such as SeMet sites, poorly occupied heavy atom sites, etc > (MR-SAD). > Or Phenix could rigid body refine (at low resolution) pseudo-atomic > models > obtained by filling in EM-models with dummy atoms. > This would help defining averaging masks and so on. > What do you think? > > Thanks in advance, > > Gino > > > *********************************************************** > Gino Cingolani, Ph.D. > Assistant Professor > Dept. of Biochemistry and Molecular Biology > SUNY Upstate Medical University > 750 E. Adams Street, Syracuse, NY, 13210 > Tel. (315) 464 8744 > Fax. (315) 464 8750 > Email: cingolag(a)upstate.edu > *********************************************************** > "Nati non foste per viver come bruti, ma per seguir virtute e > conoscenza" > ("You were not born to live like brutes, but to follow virtue and > knowledge") > Dante, The Divine Comedy (Inferno, XXVI, vv. 119-120) > _______________________________________________ > phenixbb mailing list > phenixbb(a)phenix-online.org > http://www.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 _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org http://www.phenix-online.org/mailman/listinfo/phenixbb *********************************************************** Gino Cingolani, Ph.D. Assistant Professor Dept. of Biochemistry and Molecular Biology SUNY Upstate Medical University 750 E. Adams Street, Syracuse, NY, 13210 Tel. (315) 464 8744 Fax. (315) 464 8750 Email: cingolag(a)upstate.edu *********************************************************** "Nati non foste per viver come bruti, ma per seguir virtute e conoscenza" ("You were not born to live like brutes, but to follow virtue and knowledge") Dante, The Divine Comedy (Inferno, XXVI, vv. 119-120)

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AutoMr
by Zhongli Cui 11 May '09

11 May '09

Dear all, I am using phenix automr to solve my structure. I have 2 search models with identiy of 44% and 31% respectively aligned by ClustalW. I used thiese two pdbs as search models directly in phenix, it gave a llg of about -60 and Tfz of 7. The refined structure fit the map quite well. But I wonder if the solution is OK for the negative LLG. So what should I do next? How should I prepare the search model? Shall I edit the seach models such as remove some loops or surface side chains? If I the a search model prepared by Chainsaw was used, then what is identity I have to tell phenix? Another question, I built 2 models of the same protein and of the same x-ray diffraction data with phaser in CCP4 and Phenix. In my mind, these models should be the same. But in fact they differ from each other significantly in orientation. Is there something wrong with my calculation? ---------------- Cui Zhongli Ph.D Dept. of Microbiology College of Life Sciences Nanjing Agricultural University 1 Weigang, Nanjing, 210095 Jiangsu Province P.R. China. Mail: czl(a)njau.edu.cn Tel: 86-25-84396753 Mob: (0)13951080529

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If HL coefficients are in the .mtz phenix.refine is ignoring my refinement.target= ml specification?
by Francis E Reyes 08 May '09

08 May '09

Hi all (again) It seems that despite me specifying refinement.main.target=ml in my def file, phenix.refine seems to want to keep refining against HL coefficients. It seems overtly out of the way to remove the HL coefficients definition (both the label and file) from the def file as well. Am I doing something wrong then (i.e. does it keep going back to refining experimental phases for some valid reason?) Over 80% of the ASU is built with R/Rfree in high 20's for 1.7 A data. Cheers FR --------------------------------------------- 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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changes to phenix autosol?
by Francis E Reyes 08 May '09

08 May '09

Hi all It seems that AutoSol now does some initial building when before (at least 1.3-final) it just output a phaser_x.mtz and resolve_x.mtz which would go onto phenix.autobuild after_autosol . What's this new initial building? Thanks FR --------------------------------------------- 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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Refining atoms on special positions
by Oliver Clarke 07 May '09

07 May '09

Hi, I'm running phenix.refine v1.4-3, and I'm refining a 3.3A structure that has potassium ions present on a crystallographic fourfold axis. I believe (based on previous refinement in REFMAC5.5), that these ions are at full occupancy, so I set the occupancy of each ion to 1. Despite this, there is still a lot (several 7-10sigma peaks in the Fo- Fc) of unexplained positive density, the potassium ions do not converge to their previously refined positions, and their B-factors refine upwards to values several times higher than the surrounding protein. When the same structure is refined in refmac (with ion occupancy set to 0.25), there is little to no unexplained density and the R/Rfree are substantially lower (21.9/25.9 as against 24.5/31) Does anyone have any ideas/suggestions as to what might be going on? Cheers, Oliver

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torsion_angles
by Imre Toeroe 07 May '09

07 May '09

Dear phenixers, I have seen a new strategy option, *torsion_angles* in the def files produced by recent (1.4-5x) phenix.refine. Is it active? How can one use it? Thanks, Imre

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Refining f'/f"?
by Leigh Allen 07 May '09

07 May '09

Hello, I'm new to the world of x-ray crystallography. I just solved my first SAD structure and I'm on to the refinement stage. The difference map generated by phenix.refine has really big positive peaks all around my MET residues. If I switch the atoms to MSE, then I get large negative peaks. Firstly, I'm not sure if I'm supposed to represent these residues as MET or MSE because it's a "native," high resolution (1.85) dataset, but it the protein had MSE residues. When scaling this data, I did not keep F(+) and F(-) separate. The protein's phases were generated using SAD data to 2.7A, which using SHARP led to a remarkably interpretable map that allowed me to build in the protein by hand. My second question is, how should I handle the issue with large + MET/large - MSE peaks? Do I need to rescale my data to treat it as anomalous data or is there something I can do within Phenix to fix my problem. I tried the phenix.refine GUI and set it up to refine f' and f", but it appears that nothing really changed. Thanks! Leigh Allen ******* Ph.D Candidate McCafferty Lab Department of Chemistry Duke University

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phenix.refine Ile peptide bond breaks
by SIPPEL,KATHERINE H 06 May '09

06 May '09

Hi all, I've got a 2.1 angstrom structure that I am refining. The space group is P212121. The structure is a dimer which I am refining without NCS restraints. Each monomer has 333 residues and one ligand, there are 33 Ile residues in each monomer. The first couple of rounds of refinement I had no problems, but during the third round phenix started breaking the main chain at the peptide bond and the side chain between CA and CB on some of the Ile residues. Specifically there are two breaks in chain A and four breaks in chain B, with two additional residues in chain B breaking only the CA-CB bond. Only one of the breaks is between the same residue in both chains. I'll be boring and run through my refinement process. This is the first time I've used phenix for anything but AutoSol and it is very likely that I have messed something up. Feel free to correct anything else I've done wrong along the way. Round 1, I rigid body refined chain A and B independently, performed simulated annealing, and refined individual coordinates. In COOT I manually refined and removed some bad loops. A few of the Ile were deleted but none of the problem ones Round 2, I refined individual_sites and individual_adp and turned off simulated annealing. In COOT I rebuilt some of the loops and fitted the ligand into the density. I generated a .cif file in eLBOW. Round 3, I refined the same as round 2 except that I included the .cif file. This is when I got the peptide bond breaks for Ile residues. I figured it was a fluke, fixed the breaks in COOT and rebuilt a few more residues in the loops. I ran another round of refinement and the same breaks showed up in the pdb file. I double checked the .geo file to see if Real Space Refine had made the bond distances were too long, but all the input distances were within one or two hundredths of an angstrom from ideal. I considered it was maybe the ADP refinement as I was borderline pushing the number of parameters I was refining given the number of reflections I had. I turned off the individual_adp refinement and reran refine_003. No luck. I tried increasing the weight of the geometry restraints over the observed data. I did this by increasing target_weights.wxc_scale = 1.5. (I suspect that I got this wrong. As I understood the wxc_scale is the ratio of the geometry restraints to the observed data. Feel free to point and laugh as long as you also provide an explanation as to what this parameter really means and how I should have done it,please.) Still no luck. For future information none of the problem isoleucines were located near the ligand or near any of the loops I was rebuilding. I think that's everything. Any help would be appreciated, Thanks, Kat -- SIPPEL,KATHERINE H Ph. D. candidate McKenna Lab Department of Biochemistry and Molecular Biology College of Medicine University of Florida

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