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cut out density vs FFT
by Yunji Wu 19 Feb '13

19 Feb '13

Hi all-- I'm trying to make a 2Fo-Fc map with density wrapped around my asymmetric unit to make a figure showing density for my sub-8Å structure. I am not building into this map or doing MR. My model was refined in Phenix.refine (R's in the high 30's), and so I have the output model .pdb and the output map coefficients .mtz. When I input these into cut_out_density, the only "map labels" input options it gives me are 2FOFCWT, PH2FOFCWT, which seem fine. Using either box, sphere, or model for the cutout type, I get a VERY nice looking map that looks extremely solvent flattened and tight around my model, albeit still to 8Å. Suspicious of anything that looks good, I went to try the same thing in FFT (CCP4). There I generated a simple map (using the same .mtz) to cover all atoms in a pdb file- the same model I put into cut_out_density. For the input I selected 2FOFCWT and PH2FOFCWT, leaving Sigma and Weight unassigned. The map that comes out of this looks terrible, with a lot of noise and little density corresponding to the model aside from what one might expect from a very poor resolution experimental map. I've also tried using FFT in Phenix to do the same thing, and it gives like better maps than CCP4 FFT but worse than cut_out_density. It almost appears as if there is some additional solvent flattening going on, which I know happens at the beginning of refinement, but that should be intrinsic to the map coefficients file which is common to everything I've tried. My questions are (1) what should I use for what I am trying to do (just display 2Fo-Fc density around asymmetric unit for a figure) and (2) what is the difference between what these three programs (cut_out, FFT-CCP4, FFT-Phenix) are calculating? The inputs are exactly the same -- mtz (2FOFCWT and PH2FOFCWT) and pdb output from refinement. Thanks for the help, Yunji

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Problems placing second component in Phaser
by Morten Groftehauge 19 Feb '13

19 Feb '13

Hi guys, So I have a solution that covers some of target and I ran it through phenix.mr_rosetta and I am actually kinda happy with the output from there. However, while I can place both (NCS=2) with the original model I only seem to be able to place one of the rosetta models, even with 'packing function' set to 'all' or 'allow'. Original model - Refinement after placing component 1: LLG 175 Refinement after placing component 2: LLG 620 Rosetta model (packing 'all') - Refinement after placing component 1: LLG 295 Refinement after placing component 2: LLG 390 Rosetta model (packing standard) - Refinement after placing component 1: LLG 295 Refinement after placing component 2: LLG 350 More notably there is little to no increase in TFZ with the placement of the second component... There are more residues in the Rosetta model and I would expect more clashes but this looks weird. Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University

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where is brute rotation output
by Ursula Schulze-Gahmen 18 Feb '13

18 Feb '13

Hi, Has anybody experience with the output from the brute rotation function. I ran this and it finished without complaints, but there are no output files ***.rlist or ***.sol in the directory. I cannot figure out why and how to continue now with the translation function. From the log file it seemed that there were peaks that could have been written out. Thanks for any help Ursula -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220

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Phaser problem for flexible domain of protein
by Appu kumar 18 Feb '13

18 Feb '13

Dear user, I have problem in finding the phase for the flexible DNA binding domain of LTTR protein. The unit cell parameters are ,Unit Cell: 46.92 105.30 47.75 90.00 103.26 90.00, and space-Group P 1 21 1. Molecular weight of protein is 36000Da. When i am running phaser it showing that Most probable MW of protein in asu for resolution = 49714.8. Could you please guide me how to set the proper setting for finding the correct rotation function and translation function setting to get the phases for the full length protein. I m getting the phases for the ligand binding domain but not for the DNA binding domain. Could you please suggest me some tricks which would work to get the phase. Thanks a lot in advance. Appu

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Trouble with Phenix
by Rachel North 17 Feb '13

17 Feb '13

Hi, I have been having some issues with Phenix. When I input the mtz file (generated from CCP4), an error message appears... "File format error: cctbx Error: invalid combination of values while extracting anomalous array from columns: F New, SIGF New, DANO New, SIGDANO New, hkl= -3,25,44." This happens when I try to use the reflection file editor, xtriage and phenix refine. I have tested datasets collected from other crystals in the same condition and still have the same problem. I have also tried to process my data using different programs (XDSME and iMOSFLM) before going to CCP4 and then Phenix for refinement. I have never had this problem before and I have run every program on CCP4 the same way that I usually do. Does anyone know how I fix this or where I am going wrong? Kind Regards

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how to make covelent link of BME with Cys and refine
by Rajeshwer Sankhala 15 Feb '13

15 Feb '13

Hello Nat, Thank you so much for your suggestion. I did run phenix refinement in nightly build phenix version with " intra_chain = True " command. This time refinement worked but again in out put pdb no disulfide bond is estabilished between cys and BME. I provided .pdb, .mtz and .cif files as input. Well can you please suggest me how i can provide both BME and Cys-BME (SS bond) restrains in .cif file? if that happens then i feel refinement should work. I am attaching my .cis file below please have a look and tell me if anything wrong with this? BME.cif # electronic Ligand Builder and Optimisation Workbench (eLBOW) # - a module of PHENIX version 1.8.1-1168 (Mon Sep 28 16:48:00 2012) # - file written: Sat Jan 26 17:25:31 2013 # # Input file: Ref226_154-331-VLD2-BME.pdb # Random seed: 3628800 # Residue: BME # data_comp_list loop_ _chem_comp.id _chem_comp.three_letter_code _chem_comp.name _chem_comp.group _chem_comp.number_atoms_all _chem_comp.number_atoms_nh _chem_comp.desc_level BME BME 'Unknown ' ligand 10 4 . # data_comp_BME # loop_ _chem_comp_atom.comp_id _chem_comp_atom.atom_id _chem_comp_atom.type_symbol _chem_comp_atom.type_energy _chem_comp_atom.partial_charge _chem_comp_atom.x _chem_comp_atom.y _chem_comp_atom.z BME S S S2 . -19.3229 -20.3986 17.2400 BME H5 H H . -18.1988 -20.0550 17.0819 BME C1 C CH2 . -19.3558 -21.8852 18.1928 BME H3 H HCH2 . -18.8480 -21.7572 19.0093 BME H4 H HCH2 . -18.9637 -22.6079 17.6781 BME C C CH2 . -20.7668 -22.2261 18.5333 BME H1 H HCH2 . -21.1200 -21.5643 19.1482 BME H2 H HCH2 . -20.7965 -23.1021 18.9488 BME O O OH1 . -21.5280 -22.2370 17.3822 BME H H HOH1 . -22.3435 -22.5247 17.5704 # loop_ _chem_comp_bond.comp_id _chem_comp_bond.atom_id_1 _chem_comp_bond.atom_id_2 _chem_comp_bond.type _chem_comp_bond.value_dist _chem_comp_bond.value_dist_esd BME S H5 single 1.186 0.020 BME S C1 single 1.766 0.020 BME C1 H3 single 0.970 0.020 BME C1 H4 single 0.970 0.020 BME C1 C single 1.491 0.020 BME C H1 single 0.970 0.020 BME C H2 single 0.970 0.020 BME C O single 1.380 0.020 BME O H single 0.885 0.020 # loop_ _chem_comp_angle.comp_id _chem_comp_angle.atom_id_1 _chem_comp_angle.atom_id_2 _chem_comp_angle.atom_id_3 _chem_comp_angle.value_angle _chem_comp_angle.value_angle_esd BME C1 S H5 109.48 3.000 BME C C1 H4 109.47 3.000 BME C C1 H3 109.47 3.000 BME H4 C1 H3 109.47 3.000 BME C C1 S 109.47 3.000 BME H4 C1 S 109.47 3.000 BME H3 C1 S 109.47 3.000 BME O C H2 109.47 3.000 BME O C H1 109.47 3.000 BME H2 C H1 109.47 3.000 BME O C C1 109.47 3.000 BME H2 C C1 109.47 3.000 BME H1 C C1 109.47 3.000 BME H O C 109.48 3.000 # loop_ _chem_comp_tor.comp_id _chem_comp_tor.id _chem_comp_tor.atom_id_1 _chem_comp_tor.atom_id_2 _chem_comp_tor.atom_id_3 _chem_comp_tor.atom_id_4 _chem_comp_tor.value_angle _chem_comp_tor.value_angle_esd _chem_comp_tor.period BME Var_01 O C C1 S 52.25 30.0 3 BME Var_02 H1 C C1 S -67.75 30.0 3 BME Var_03 H2 C C1 S 172.25 30.0 3 BME Var_04 O C C1 H3 172.25 30.0 3 BME Var_05 O C C1 H4 -67.75 30.0 3 # loop_ _chem_link.id _chem_link.comp_id_1 _chem_link.mod_id_1 _chem_link.group_comp_1 _chem_link.comp_id_2 _chem_link.mod_id_2 _chem_link.group_comp_2 _chem_link.name CYS-BME CYS DEL-HG . BME DEL-HS . SS-bridge # Thanks

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HETATM automated chain assignment
by Talon Romain 15 Feb '13

15 Feb '13

Hello to the Phenix bulletin board community, I would like to know if I could find a tool to automatically assign HETATM atom (or even, water molecules) to the nearest protein chain ? In my case, I have 4 protein chains in the asymmetric unit : A, B, C and D. I would like to assign each ions and each ligands (which are numerous) with the chain letter of the nearest residue that coordinate them. Usually, I rename everything by hand but as the in-house program of the PDBe AutoDep deposition tool automatically do that... I beg your pardon if this question has just been posted here. I didn't find any tool either in the Phenix Suite or in the "Extensions" and "Calculate" menus of the Coot program (v0.7). Best regards. Romain Talon

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how to make covelent link of BME with Cys and refine
by Rajeshwer Sankhala 13 Feb '13

13 Feb '13

Hello Sabine Yah i could specify link in the pdb and also could see it in coot but when i go for refinement then bond is broken. I given pdb mtz and cif files in refinement. The only problem i feel could be that i don't have restrains for SS bond in cif file but i don't know how to generate SS-bond restrains. Thanks RAJ

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how to make covelent link of BME with Cys and refine
by Rajeshwer Sankhala 13 Feb '13

13 Feb '13

Thanks Nigel, I am using phenix 1168 but when i type intra_chain = True command with phenix.refine it gives error message as "intra_chain = True is unknown command". The actual command i used was " phenix.refine .pdb .mtz .cif intra_chain = True". can you please explain how to resolve it. Raj

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Re: [phenixbb] how to make covelent link of BME with Cys and refine
by Ed. Pozharski 13 Feb '13

13 Feb '13

Excellent point. The difference between using bme and cme is rather ideological. I would say that incorporating cme implies that your protein was somehow synthesized with non-canonical amino acid. Usind bme seems more in line with what actually happens - betamercaptoethanol forming disulfide bond with a cysteine. In practical sense, both things work. Until IUCr comes up with a convention, one is free to ignore ideological considerations :) -------- Original message -------- From: Abhinav Kumar <abhinavk(a)slac.stanford.edu> Date: To: phenixbb(a)phenix-online.org Subject: Re: [phenixbb] how to make covelent link of BME with Cys and refine How about using residue CME which is the CYS and BME linked together. CME is included in the CCP4 monomer library, and so Phenix should accept it without a dictionary file. Thanks, Abhinav JCSG@SSRL, SLAC (650) 926-2992 On 02/13/2013 01:02 PM, Nigel Moriarty wrote: Raj In the more recent versions of PHENIX, phenix.refine can found and link atoms closer than 3 angstrom if you specify intra_chain=True. In the older versions, you can use phenix.ligand_linking to generate the needed files. Nigel PS PHENIX does not read SSBOND or LINK records but there is a script to generate the link files from the LINK records. On Tue, Feb 12, 2013 at 11:56 PM, Sabine Schneider <sabine.schneider(a)mytum.de> wrote: Hello Raj, do you have the link specified in you PDB file? Here is an example for an disulphide bond. Usually the link is the line above the CRYST1 card. SSBOND 1 CYS A 123 CYS A 123 1555 8555 2.12 Best, Sabine On 02/12/2013 11:21 PM, Rajeshwer Sankhala wrote: Hello, I can see additional density just next to Cys residues in my protein structure and it looks like BME is covalently linked to cys. I could form a disulfide bond between BME and Cys in "Refmac 5 review restrain" but when i subject it for refinement BME is thrown away (no disulfide bond). I also given Cif file in refinement but it didnot help. I have a feel that i am missing something in cif file. Can anyone suggest step by step starting from adding BME to making a link with BME (Cys-BME) and then refinement? Raj _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org http://phenix-online.org/mailman/listinfo/phenixbb -- Nigel W. Moriarty Building 64R0246B, Physical Biosciences Division Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 Email : NWMoriarty(a)LBL.gov Fax : 510-486-5909 Web : CCI.LBL.gov

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