- phenixbb - phenix-online.org

Re: [phenixbb] need help for crystallization
by Kevin Jin 30 Mar '12

30 Mar '12

My pleasure ! Since this may be help for other people, I also cc it to CCP4BBS. According to your email, I guess the buffer is phosphate buffer at pH 7.4. You can do a quick buffer exchange before crystallization. Since PO4 is a competitor in this case, I will also avoid PO4 and Cocodylate buffer from screen kits. Here is what you can do, 1. concentrate your protein using an amicro centrocon with 3KD cutoff. 2. Measure how much buffer has been spin down, then add equal amount of Tris with same pH back to the top. 3. Then, spin down again. 4. Repeat several times, 5 times may be good enough. 5. Most of Phosphate buffer should be removed. That's what I used before. Let me know if it works. Best, Kevin Jin On Fri, Mar 30, 2012 at 9:40 AM, Afshan Begum <afshan490(a)yahoo.com> wrote: > > Dear Kevin > > I have seen your website and come to you to discuss my problem actually i > want to co-crystallized my enzyme with inhibitor but problem is that the > phosphate ion come to the active site and occupied the cavity and my > inhibitor not bind to the active center i purchase this enzyme from a > commercial source and they purify with phosphate buffer that's why also > phosphate is occupied the cavity how can i get ride of it from the active > center could you kindly help me regarding this i would be really thankful to > you. > > > Best Regards > > AFSHAN > =========================================== > Dr. Afshan Begum > University of Hamburg > Institute of Biochemistry and Molecularbiology > Laboratory for Structural Biology of Infection and Imflammation > c/o DESY, Build. 22a > Notkestr. 85 > 22603 Hamburg > Germany > Home phone: +49 40 22888618 > Fax: +49 40 8998-4747 > E-mail: afshan490(a)yahoo.com > -- Kevin Jin Sharing knowledge each other is always very joyful...... Website: http://www.jinkai.org/

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Re: [phenixbb] phaser_ep
by Airlie McCoy 30 Mar '12

30 Mar '12

Dear Kay, You can control the distance for the atom separation/anisotropy in Phaser with the llgcomplete.clash input parameter. By default llgcomplete.clash is zero, which means Phaser will calculate a value based on the resolution. If a value is set this will override the default. Apologies, this was not explained in the wiki, but is now! Airlie

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phaser_ep
by Kay Diederichs 30 Mar '12

30 Mar '12

Dear all, is there a way to modify the decision constants in phaser_ep? Specifically, I want to refine against heavy-atom derivative with one strong site. The resolution of the data is 3.6A; the substructure (4 sites) was solved at 8A. Upon refinement, phaser finds two more sites at distances of ~3.5A to the strongest site. Furthermore, it finds two holes at distances of ~3.5A to the strongest site. Now I would like phaser to treat the site as anisotropic. However, the distances are beyond its decision constant (~2.54A), so it chooses to add two sites to my HA model, but they refine poorly. How is the decision constant calculated? Can I influence the calculation? I found nothing in the phaser wiki. Alternatively: how can I tell phaser_ep that it should refine a given input atom anisotropically? I tried to add a ANISOU line to the PDB file but the program seems to ignore it. thanks, Kay -- Kay Diederichs http://strucbio.biologie.uni-konstanz.de email: Kay.Diederichs(a)uni-konstanz.de Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature "smime.p7s".

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A small trick to make cover glass extremely hydrophilic for hanging drop
by Kevin Jin 29 Mar '12

29 Mar '12

Dear All, Making the surface of cover glass hydrophilic could let drop with organic solvent (MPD, etc) shrink into a ball shape, not spread out. Here is the method I used before. http://www.jinkai.org/Xtal.html Kevin -- Kevin Jin Sharing knowledge each other is always very joyful...... Website: http://www.jinkai.org/

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a strategy for endotoxin removal
by Kevin Jin 29 Mar '12

29 Mar '12

Dear All, I developed a method to remove endotoxin before and it worked pretty well. Here is the strategy for this protocol. http://www.jinkai.org/Endotoxin.html I hope it is useful. Best, Kevin -- Kevin Jin Sharing knowledge each other is always very joyful...... Website: http://www.jinkai.org/

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reference model restraints now support multiple reference model files
by Jeff Headd 28 Mar '12

28 Mar '12

Hi PHENIX users, After numerous user requests, we've added support to phenix.refine for multiple reference model input files. If you are refining a complex and have reference models for individual components, you may now input as many files as necessary. A parameter file would include: refinement { reference_model { file = "reference_A.pdb" file = "reference_B.pdb" } } Chain matching is still carried out automatically, so simply specifying the file paths is usually enough for the restraints to be generated for refinement. Details of chain matches are printed in the .log file, as well as in the reference_model scope in the .eff file. If anyone has questions, please let me know. Thanks! Jeff

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positive density around heavy metals
by Subhani Bandara 28 Mar '12

28 Mar '12

Hi everyone, I have refined a protein with a metal-chelate complex at 1.4 resolution and saw positive density around some metals that are on a two fold. I refined those chelator complexes with fixed occupancy to avoid the negative density seen around it as they are partially occupied. Also I had to avoid xyz refinement after fixing them on special position, to avoid moving away from two fold. I have attached a figure of one of those complexes. If I increase occupancy then it shows negative density. How can I get rid of this positive density. Thanks in advance for the help Subhani

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Re: [phenixbb] merging in hires data in AutoBuild
by Desiree Ho-Chi Fong, Ms 27 Mar '12

27 Mar '12

It works! Thank you. Desiree

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Generating a list of peak heights in a Bijvoet difference Fourier
by John Rose 27 Mar '12

27 Mar '12

Hi, Is there a way to get a print out of the peak positions and their heights from a Bijvoet difference Fourier map? I can generate the map and see the peals but I would like a listing also. Thanks, John Rose John Rose Ph.D. Associate Professor B204B, The Fred C. Davison Life Sciences Complex 120 Green Street Department of Biochemistry and Molecular Biology University of Georgia Athens, GA 30602-7229 ========================================= Phone: 706-542-1750 Fax: 706-542-3077

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merging in hires data in AutoBuild
by Desiree Ho-Chi Fong, Ms 26 Mar '12

26 Mar '12

Hello all, I am using PHENIX 1.7.3-928 and I am trying to merge a high resolution native data set with a MAD data set in AutoBuild. I have run AutoBuild through the GUI as well as on the command line. Here is the command I used: phenix.autobuild data=AutoSol_run_1_/exptl_fobs_phases_freeR_flags_1.mtz hires_file=hires.mtz model=AutoSol_run_1_/overall_best.pdb rebuld_in_place=True The data file, exptl_fobs_phases_freeR_flags.mtz from AutoBuild only includes data to the resolution of the MAD data set. The higher resolution native data was not incorporated into this mtz file. Looking at the log file, the hires data was merged with expel_fobs_phases_freeR_flags_1.mtz from AutoSol (with AutoBuild after) and followed by anisotropy correction. This was then used for density modification. However, the high resolution data was never included in the mtz file for refinement in AutoBuild. My understanding is that if hires data is specified in AutoBuild, the phases would be extended and included in map calculation as well as subsequent refinement. The output exptl_fobs_phases_freeR_flags.mtz from AutoBuild would also include the hires data for subsequent refinement. Is this correct? Thank you for your help. Desiree

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R value difference in model vs data vs refinement
by Christian Roth 23 Mar '12

23 Mar '12

Dear all, I refined a structure in phenix quite good resolution (1.4 Ang.) After the last refinement I created the "table 1" to extract all data. Interestingly I got a warning due to small changes in the R values R/Rfree after Refinement. 15,46/18,16 Phenix modelvs.data gives me R/Rfree 15,34/18,07 Interestingly this is nearly exactly the value which I got after the first OHS scattering optimization from phenix R/Rfree 15,43/18,03 The differences are of course very small and negeligible, but I wonder why it happens. Esspecially taking into account that it was refined with phenix and in this case I would expect that both values match exactly. Thank you very much in advance Best Regards Christian

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sequence file for DNA-Protein complex
by Sita Ram Meena 23 Mar '12

23 Mar '12

Hello Everyone. As per my knowledge we gives the Protein sequence .SEQ OR .DAT file format having only protein sequence in Caps and no gaps now, Please suggest me how we can give a sequence file for double stranded DNA-Protein Complex for the use in Phenix autobuild. Thank you. Best Regards Sita Ram

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error generating output file overal_best pdb
by Andres PALENCIA 23 Mar '12

23 Mar '12

Dear Phenix users, I have been running Phenix_Autobuild successfully for the last houra. The program generates the corresponding Autobuild_run_** folders as usually, however I got an error message when the program was trying to generate the overal_best.pdb file after the Autobuild cycle 3. I copy below the error message that I got, thank you in advance for any help: OSError : [Errno 2] No such file or directory: '/user/palencia' Traceback (most recent call last): File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/phenix/wxGUI2/Home/Jobs.py", line 170, in OnTimer self.project.refresh_detached_jobs() File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/phenix/wxGUI2/Tracking.py", line 72, in refresh_detached_jobs tmp_dir = self.get_tmp_dir() File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/phenix/phenix/tracking/project.py", line 97, in get_tmp_dir os.makedirs(tmp_dir) File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/build/intel-linux-2.6-x86_64/base/lib/python2.7/os.py", line 150, in makedirs makedirs(head, mode) File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/build/intel-linux-2.6-x86_64/base/lib/python2.7/os.py", line 150, in makedirs makedirs(head, mode) File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/build/intel-linux-2.6-x86_64/base/lib/python2.7/os.py", line 150, in makedirs makedirs(head, mode) File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/build/intel-linux-2.6-x86_64/base/lib/python2.7/os.py", line 150, in makedirs makedirs(head, mode) File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/build/intel-linux-2.6-x86_64/base/lib/python2.7/os.py", line 150, in makedirs makedirs(head, mode) File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/build/intel-linux-2.6-x86_64/base/lib/python2.7/os.py", line 150, in makedirs makedirs(head, mode) File "/softdist/exp/pxsoft/phenix/v1.7.3-928/redhate4-x86_64/phenix-1.7.3-928/build/intel-linux-2.6-x86_64/base/lib/python2.7/os.py", line 157, in makedirs mkdir(name, mode) OSError: [Errno 2] No such file or directory: '/user/palencia' Platform info: __FILE__ = /net/anaconda/scratch1/phenix/phenix-1.7.3-928/cctbx_project/boost_adaptbx/meta_ext.cpp __DATE__ = Dec 6 2011 __TIME__ = 04:18:10 __x86_64__ __linux __GNUC__ = 3 __GNUC_MINOR__ = 4 __GNUC_PATCHLEVEL__ = 2 boost::python::cxxabi_cxa_demangle_is_broken(): false __GXX_WEAK__ = 1 __LP64__ = 1 __VERSION__ = 3.4.2 20041017 (Red Hat 3.4.2-6.fc3) FE_INEXACT = 32 FE_DIVBYZERO = 4 FE_UNDERFLOW = 16 FE_OVERFLOW = 8 FE_INVALID = 1 FE_ALL_EXCEPT = 61 __SSE2__ = 1 BOOST_LIB_VERSION = 1_48 PY_VERSION = 2.7.2 PYTHON_API_VERSION = 1013 sizeof(bool) = 1 sizeof(short) = 2 sizeof(int) = 4 sizeof(long) = 8 sizeof(std::size_t) = 8 sizeof(void*) = 8 sizeof(long long) = 8 sizeof(float) = 4 sizeof(double) = 8 sizeof(long double) = 16 sizeof(boost::int32_t) = 4 sizeof(boost::uint32_t) = 4 sizeof(wchar_t) = 4 __PTRDIFF_TYPE__ Py_USING_UNICODE sizeof(PY_UNICODE_TYPE) = 2 gnu libc version: 2.5 BOOST_ADAPTBX_TYPE_ID_SIZE_T_EQ_UNSIGNED_LONG os.name: posix sys.platform: linux2 sys.byteorder: little platform.platform(): Linux-2.6.18-238.9.1.el5-x86_64-with-redhat-5.7-Final platform.architecture(): ('64bit', 'ELF') floating_point_exceptions.division_by_zero_trapped: False floating_point_exceptions.invalid_trapped: False floating_point_exceptions.overflow_trapped: False number of processors: 24 Memory total: 25,263,722,496 Memory free: 20,157,317,120 import thread: OK "hello" = ['h', 'e', 'l', 'l', 'o'] u"hello" = ['h', '\x00', 'e', '\x00', 'l', '\x00', 'l', '\x00', 'o', '\x00'] u"\u00C5" = ['\xc5', '\x00'] as utf-8 = ['\xc3', '\x85'] LATIN CAPITAL LETTER A WITH RING ABOVE = Ã

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Rosetta and disulphides
by Morten Groftehauge 23 Mar '12

23 Mar '12

Hi phenix-bb'ers, Is there anyway to make phenix.mr_rosetta take disulphide bridges into account? It's a rather enormous restraint on conformational space after all (wait is that a constraint or restraint? I get confused). Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University

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about readyset
by Ming 23 Mar '12

23 Mar '12

Dear all, I was using ready set to get my pdb ready. It automatically built the ligand cif file from scratch too. And the phenix.refine was fine with the files from ready set. Then I tried another method in which I threw everything that phenix.refine needs, including pdb file, ligand cif file, ligand pdb file, mtz. And phenix.refine ran smoothly again. But the refinement results were different between these two ways. The R fac is 1% different. I think ready set is only making the phenix.refine easier by generating ligand and building H, but if I do all the work of ready set and prepare the files for phenix.refine. Aren't they supposed to give the same results back? Can anyone help me here? Thanks a lot. -- Ming

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Partially reduced disulfides
by Christopher Wanty 22 Mar '12

22 Mar '12

Hi, I have a structure that appears to have a combination of reduced and oxidised disulfides. >From previous reading on the board I've taken the strategy of dropping the disulfide detection cutoff to 0.1 then added back the disulfides as appropriate using restraints. The problem I now have is that for some of these cysteine pairs there are clearly oxidised and reduced positions in the density. Modelling altloc's for each of these works, but the occupancies do not refine as I expected; the altloc's involved in the disulfide I expected to have the same occupancy as their counterpart in the disulfide. To try to get these to match, I introduced occupancy constraint that look like: constrained_group { selection = (chain A and resseq 276 and altloc B) or (chain A and resseq 286 and altloc B) } But with that the sum of occupancies for altloc 276 (for example) is not adding to 1.00. My thought is that I need to change the occupancy constraints but I'm not sure how to make them do what I want. Any help/advice is appreciated. Regards, Chris ------------------------------------------------------- Dr. Christopher Wanty christopher.wanty(a)uwa.edu.au Research Associate Biomolecular, Biomedical and Chemical Sciences, Building M310 University of Western Australia 35 Stirling Highway Crawley 6009 Western Australia Australia

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Documentation in last Dev versions
by Alexander Batyuk 22 Mar '12

22 Mar '12

Dear developers, Is there any particular reason for missing documentation file in phenix-dev-1008 and up (it could not be found on the website as well)? dev-1005 is fine. Best wishes, Alex -- Alex Batyuk The Plueckthun Lab www.bioc.uzh.ch/plueckthun

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Hydrogens
by Christopher Wanty 21 Mar '12

21 Mar '12

Hi all, I've been refining with riding hydrogens, and from what I've read hydrogen contribution should be included in Fo calculation automatically for maps. What I'm seeing after refinement is a lot of little bits of green in the difference density that look like they could be explained by hydrogens. I'm working at 1.4A, attached image gives an example and shows contours at 3 sigma for difference map. Is there a setting/parameter I need to adjust to get this to work properly? Kind Regards, Chris ------------------------------------------------------- Dr. Christopher Wanty christopher.wanty(a)uwa.edu.au Research Associate Biomolecular, Biomedical and Chemical Sciences, Building M310 University of Western Australia 35 Stirling Highway Crawley 6009 Western Australia Australia

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building a peptide-like polymer with phenix.elbow
by Geoffrey Feld 21 Mar '12

21 Mar '12

Greetings, I have a co-crystal structure of a protein bound to an interesting polymer -- poly-D-g-glutamic acid (poly glutamic acid with D chirality and gamma linked, rather than alpha linked). In the structure, I have density corresponding to different links, from 1 to 6 (so far). I'd like to build a ligand that has "residues" much like a polypeptide polymer so that I can refine each residue individually (hopefully in COOT). Up till now, I've been using SMILES to generate the different length polymers so I have pdbs and cif files, and then (painstakingly) modelling them into COOT, building restraints for refinement, etc. More recently, I used maestro to build a dimer that at least has the right atom labels you'd expect for a protein. However, I can't use phenix now to generate cif files because it is confused by the GLU. Is there a way to use phenix.elbow to make a multi-residue polymer? I'm also doing some monte carlo minimizations in macromodel to generate models of the various low energy states and would like to use them as building blocks, so an easy order-of-operations would be to generate the files in macromodel and feed them in via pdbs to phenix.elbow to generate cif restraints. Any help on this would be greatly appreciated. Cheers, Geoff -- Geoffrey K. Feld Department of Chemistry 492 Stanley Hall University of California, Berkeley "Vigilia pretium libertatis"

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a small trick for SDS-Gel storage
by Kevin Jin 20 Mar '12

20 Mar '12

Dear All, Maybe most of you already knew. I just put my understanding of SDS page gel storage here, if you want to make gel yourself and safe some money. http://www.jinkai.org/SDS_page.html If I am wrong, please let me know. Sharing knowledge each other is always joyful. -- Kevin Jin Protein crystallographer and Biopolymer chemist Personal Website: http://www.jinkai.org/

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A possible conjugative relaxase with novel fold for DNA cleavage?
by Kevin Jin 20 Mar '12

20 Mar '12

Dear All, The protein structure of Jann_2411 (PDB 3H0N) from Jannaschia sp. strain CCS1 carries a novel fold for unknown function. Zn-finger is indentified in the C-terminus. However, the N-terminus with a metal binding site presents a novel fold. As the person who solved and characterized this protein, I would like to put some further observation here for your consideration. My analysis is available here: http://www.jinkai.org/Novel_fold_3H0N.html Best regards, Kevin -- Kevin Jin Protein crystallographer and Biopolymer chemist Personal Website: http://www.jinkai.org/

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NCS operators
by Melania Strycharska 19 Mar '12

19 Mar '12

Dear all, I have a bit of a problem applying NCS to my fairly low resolution structure. If I try to look for NCS symmetry automatically - phenix doesn't find any. If I specify sections that I want to be related by NCS, the script usually crashes. I have a dimer in the asymmetric unit, however it appears that one domain is related by 3-fold symmetry while the other is related by 6 fold symmetry. I can "cheat" phenix and generate operators for each of the domains by feeding a stripped pdb file (one that contains only a dimer of one domain or the other) to phenix.simple_ncs_from_pdb and then it easily identifies the ncs operators... unfortunately the simple_ncs_from_pdb.ncs file generated that is meant to be fed into phenix.refine contains only the chain selections, not the actual operators. Is there a way to specify different NCS operators (rotational matrix and translational vector) for specific chain selections in the .eff file or under GUI? or if there isn't, how can I still take advantage of my NCS symmetry? Also, is there any obvious reason why NCS operators that I generated with Phenix do not match the operators that I found using CCP4 Lsqkab? (yes I already accounted for chain A moving onto B or chain B moving onto A) Can I choose to apply NCS symmetry to just my backbone? How would I specify that? thanks so much! Melania

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GUI parameter files
by Leonid Sazanov 19 Mar '12

19 Mar '12

Hi, I have a following problem in dev-874 version: If I restore a previous job in refine GUI, to run it with modified settings, and include a new parameter file .eff with definitinon of secondary structure restrains, these new restrains are combined with previously used restrains. How do i get rid of previous restrains? Older file is not visible in inputs, but its settings are still used. And in general, how can one be sure which parameters are used at the start of each refine run? If older settings loaded from .eff or .param file are not visible, the only way is to look through new .eff file, which takes a lot of time. Has this been solved in latest builds perhaps? Thank you, Leo -- Dr. Leonid A. Sazanov Research group leader Medical Research Council Mitochondrial Biology Unit Wellcome Trust / MRC Building Hills Road Cambridge CB2 0XY WEB: www.mrc-mbu.cam.ac.uk Tel: +44-1223-252910 Fax: +44-1223-252915

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Refinement using data with pseudo translational symmetry
by Bret Wallace 18 Mar '12

18 Mar '12

To all, First I apologize for the long email, but I wont to make sure that I give enough information to describe the problem. I have been working on a structure of two proteins in complex with each other (one forms a homodimer, with each monomer bound to 1 monomer of the other protein), using data, that according to phenix.xtriage contains pseudo translational symmetry (output pasted below). I have done a lot of searching of both the phenixBB and ccp4BB regarding solutions to this problem, but unfortunately most responses seem to be directed at resolving issues with MR. I have successfully performed MR using both phenix phaser, ccp4 phaser (the most updated version), as well as molrep. The issue arises upon structure refinement, where my Rwork and Rfree are essentially stuck at 28 and 33, respectively. I have built the entire structure, including ligands, except for any solvent molecules. Here are the details for the data/structure: SG: P212121 Cell: 72 124 175 90 90 90 Res: 50-2.8 Patterson analyses ------------------ Largest Patterson peak with length larger than 15 Angstrom Frac. coord. : 0.155 0.000 0.500 Distance to origin : 87.222 Height (origin=100) : 34.517 p_value(height) : 6.739e-04 The reported p_value has the following meaning: The probability that a peak of the specified height or larger is found in a Patterson function of a macro molecule that does not have any translational pseudo symmetry is equal to 6.739e-04. p_values smaller than 0.05 might indicate weak translational pseudo symmetry, or the self vector of a large anomalous scatterer such as Hg, whereas values smaller than 1e-3 are a very strong indication for the presence of translational pseudo symmetry. Xtriage notes that the if the PTS is crystallographic, that C2221 is a possible SG (x+1/6, y, z+1/2). However, neither XDS or HKL picks orthorhombic C, and the indexing/integrating doesn't work if I force it. I should also not there is no twinning detected by either xtriage or twinning servers. I have done a number of things to try to resolve this: -rescaling in lower symmetry (P21 and P1) -rescaling in the PG P222 and letting the MR program decide the proper space group -shifting origin and re-refining -a number of different refinement protocols (TLS, optimized weights/adp, simulated annealing, several cycles of rigid body, etc.) I have used HKL2000 and XDS to index/integrate/scale the data, both yield the same results, with slightly different completeness and Rmerge values, but refining with either gives similar R factor values. Does any know of any other possible things I should try to refine this data? I am happy to provide additional information upon request. Thanks in advance for any help! Bret

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selectively remove bond length restraints on atoms
by Maxime Cuypers 15 Mar '12

15 Mar '12

hello, What is the best way to selectively remove bond length restraints on some selected atoms for phenix.refine? i found the commands for a .param file: refinement { geometry_restraints.remove { angles = None dihedrals = None chiralities = None planarities = None } } but no bond length command in the manual online. http://www.phenix-online.org/version_docs/dev-283/refinement.htm otherwise there is the commands refinement { geometry_restraints.edit { bond { action= *delete change atom_selection_1= (selection1 here) atom_selection_2= (selection2 here) distance_ideal= None } } } is it a good way to deal with the problem ? thanks in advance for your help. Maxime

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