- 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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