[ccp4bb] .raw file from SAINT to pointless
Dear all, I have collected 2 datasets (space group P1, resolution 1.4) on a bruker machine using Proteum2 v2014.9-0 software. I integrated both datasets with SAINT and would like to continue with Pointless - SCALA - Truncate to finally get*scaled but unmerged data* to be able to calculate CC*, CCanom, . Pointless should be able to read .raw data (documentation) andhttp://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Bruker_data. However it fails with the error message Release Date: 6th January 2014 ** ** * POINTLESS * * 1.8.17 * ** * Determine Laue group from unmerged intensities * * Phil Evans MRC LMB, Cambridge * * Uses cctbx routines by Ralf Grosse-Kunstleve et al.* ** ** Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: /home/georg/ccp4/ccp4-6.4.0/lib/data/syminfo.lib *** * Information from CCP4Interface script *** The program run with command: /home/georg/ccp4/ccp4-6.4.0/bin/pointless has failed with error message pointless: /home/marcin/series-64-dt/checkout/pointless/sca_unmerge.cpp:1201: std::vector SCAIO::SaintRun::MakeBatch(const float&): Assertion `BHeader.num > 0' failed. *** #CCP4I TERMINATION STATUS 0 "pointless: /home/marcin/series-64-dt/checkout/pointless/sca_unmerge.cpp:1201: std::vector SCAIO::SaintRun::MakeBatch(const float&): Assertion `BHeader.num > 0' failed." #CCP4I TERMINATION TIME 26 Dec 2014 23:15:29 #CCP4I MESSAGE Task failed The "strange" thing is that it works with an insulin dataset (using Input reflection filetyp XDS) and that in the Input reflection filetyp one can just choose MTZ, XDS or scalepack, and not SAINT (raw). I would be grateful if somebody could point me in the direction where the problem is or show me an alternative route. Best regards Georg. -- Mlynek Georg University of Vienna Department of Computational and Structural Biology Max F. Perutz Laboratories Campus Vienna Biocenter 5 level -2 1030 Vienna Austria e-mail: georg.mly...@univie.ac.at mobil: +43 660 42 195 07 office: +43-1-4277-52263
[ccp4bb] capillary counterdiffusion agarose plug
Dear Colleagues, After reading a few papers about growing suitable crystals for neutron diffraction. I will do capillary counterdiffusion with an agarose plug between mother liquor and protein solution like described in http://www.ncbi.nlm.nih.gov/pubmed/23192028. However I looked quite some time now, to find how to put the 100 ul 1% low-melting agarose plug in the middle of the capillary, but was not successful. Does one use a gel loading tip or is there a better way to do this? Thanks in advance for any tips and tricks, best regards, Georg.
Re: [ccp4bb] capillary counterdiffusion agarose plug
Hi Georg, I haven't used the agarose method, but have sealed many capillaries using bees wax without an issue. Simply heat the wax until it melts, dip the capillary (rotate if it has a larger diameter) then let cool. I hope that helps, cheers! Sean Seaver, PhD P212121 http://store.p212121.com/
Re: [ccp4bb] .raw file from SAINT to pointless
Dear Georg, Since you collected your data on a Bruker machine and integrated them with SAINT you should simply scale the .raw files with the Bruker program SADABS and then read them into into XPREP. This can scale the two datasets together and produce either merged or unmerged but scaled data. The current version calculates the various correlation coefficients as a function of resolution and can display them graphically. Since you have two separate datasets, you should also calculate the CC between them. XPREP can also prepare the files for experimental phasing with shelxc/d/e. If you need further help you may contact me directly. Best wishes, George On 01/05/2015 06:07 PM, Georg Mlynek wrote: Dear all, I have collected 2 datasets (space group P1, resolution 1.4) on a bruker machine using Proteum2 v2014.9-0 software. I integrated both datasets with SAINT and would like to continue with Pointless - SCALA - Truncate to finally get*scaled but unmerged data* to be able to calculate CC*, CCanom, . Pointless should be able to read .raw data (documentation) andhttp://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Bruker_data. However it fails with the error message Release Date: 6th January 2014 ** ** * POINTLESS * * 1.8.17 * ** * Determine Laue group from unmerged intensities * * Phil Evans MRC LMB, Cambridge * * Uses cctbx routines by Ralf Grosse-Kunstleve et al.* ** ** Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: /home/georg/ccp4/ccp4-6.4.0/lib/data/syminfo.lib *** * Information from CCP4Interface script *** The program run with command: /home/georg/ccp4/ccp4-6.4.0/bin/pointless has failed with error message pointless: /home/marcin/series-64-dt/checkout/pointless/sca_unmerge.cpp:1201: std::vector SCAIO::SaintRun::MakeBatch(const float&): Assertion `BHeader.num> 0' failed. *** #CCP4I TERMINATION STATUS 0 "pointless: /home/marcin/series-64-dt/checkout/pointless/sca_unmerge.cpp:1201: std::vector SCAIO::SaintRun::MakeBatch(const float&): Assertion `BHeader.num> 0' failed." #CCP4I TERMINATION TIME 26 Dec 2014 23:15:29 #CCP4I MESSAGE Task failed The "strange" thing is that it works with an insulin dataset (using Input reflection filetyp XDS) and that in the Input reflection filetyp one can just choose MTZ, XDS or scalepack, and not SAINT (raw). I would be grateful if somebody could point me in the direction where the problem is or show me an alternative route. Best regards Georg. -- Mlynek Georg University of Vienna Department of Computational and Structural Biology Max F. Perutz Laboratories Campus Vienna Biocenter 5 level -2 1030 Vienna Austria e-mail:georg.mly...@univie.ac.at mobil: +43 660 42 195 07 office: +43-1-4277-52263 -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
Re: [ccp4bb] capillary counterdiffusion agarose plug
https://hamptonresearch.com/product_detail.aspx?cid=10&sid=65&pid=109 Granada box will work for you Thaumatin can be grown to large single crystals suitable size On Mon, Jan 5, 2015 at 9:25 AM, Georg Mlynek wrote: > Dear Colleagues, > > After reading a few papers about growing suitable crystals for neutron > diffraction. I will do capillary counterdiffusion with an agarose plug > between mother liquor and protein solution like described in > http://www.ncbi.nlm.nih.gov/pubmed/23192028. > > However I looked quite some time now, to find how to put the 100 ul 1% > low-melting agarose plug in the middle of the capillary, but was not > successful. Does one use a gel loading tip or is there a better way to do > this? > > Thanks in advance for any tips and tricks, > > best regards, Georg. > -- P
Re: [ccp4bb] .raw file from SAINT to pointless
As George says, you can do it all with Bruker programs However, Pointless is supposed to read .raw files, but when I wrote the code (at the request of a user) I didn't have complete information, so I may have missed something. The error message is from somehow getting a zero or negative batch number, and I can't immediately see how that would happen. Georg, could you send me the .raw file (gzipped or maybe just part of it if it is large), and I'll try to fix it? I note that you are running quite an old version of Pointless (CCP4 6.5 has version 1.9.23) though I don't know that this code has changed since then. Also you should use Aimless rather than Scala best wishes Phil On 5 Jan 2015, at 17:07, Georg Mlynek wrote: > > >> Dear all, I have collected 2 datasets (space group P1, resolution 1.4) on a >> bruker machine using Proteum2 v2014.9-0 software. I integrated both datasets >> with SAINT and would like to continue with Pointless - SCALA - Truncate to >> finally get scaled but unmerged data >> to be able to calculate CC*, CCanom, . >> >> Pointless should be able to read .raw data (documentation) and >> http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Bruker_data >> . However it fails with the error message >> >> >> Release Date: 6th January 2014 >> >> ** >> ** >> * POINTLESS * >> * 1.8.17 * >> ** >> * Determine Laue group from unmerged intensities * >> * Phil Evans MRC LMB, Cambridge * >> * Uses cctbx routines by Ralf Grosse-Kunstleve et al.* >> ** >> ** >> >> Spacegroup information obtained from library file: >> Logical Name: SYMINFO Filename: >> /home/georg/ccp4/ccp4-6.4.0/lib/data/syminfo.lib >> >> *** >> * Information from CCP4Interface script >> *** >> The program run with command: /home/georg/ccp4/ccp4-6.4.0/bin/pointless >> has failed with error message >> pointless: >> /home/marcin/series-64-dt/checkout/pointless/sca_unmerge.cpp:1201: >> std::vector SCAIO::SaintRun::MakeBatch(const float&): Assertion `BHeader.num >> > 0' failed. >> *** >> >> #CCP4I TERMINATION STATUS 0 "pointless: >> /home/marcin/series-64-dt/checkout/pointless/sca_unmerge.cpp:1201: >> std::vector SCAIO::SaintRun::MakeBatch(const float&): Assertion `BHeader.num >> > 0' failed." >> #CCP4I TERMINATION TIME 26 Dec 2014 23:15:29 >> #CCP4I MESSAGE Task failed >> >> >> >> >> The >> "strange" thing is that it works with an insulin dataset (using Input >> reflection filetyp XDS) and that in the Input reflection filetyp one can >> just choose MTZ, XDS or scalepack, and not SAINT (raw). >> >> I would be grateful if somebody could point me in the direction where the >> problem is or show me an alternative route. >> >> Best regards Georg. >> >> >> >> > > -- > Mlynek Georg > University of Vienna > Department of Computational and Structural Biology Max F. Perutz Laboratories > Campus Vienna Biocenter 5 level -2 > 1030 Vienna Austria > > e-mail: > georg.mly...@univie.ac.at > > mobil: +43 660 42 195 07 > office: +43-1-4277-52263 >
Re: [ccp4bb] capillary counterdiffusion agarose plug
Hi, Warmed up capillary and hamilton syringe works fine (i used heat lamps similar to pizza heaters used by all-night pizza joints). You can get a cheap heat lamp from most pet stores (terrarium heaters). Artem On Jan 5, 2015 11:32 AM, "Georg Mlynek" wrote: > Dear Colleagues, > > After reading a few papers about growing suitable crystals for neutron > diffraction. I will do capillary counterdiffusion with an agarose plug > between mother liquor and protein solution like described in > http://www.ncbi.nlm.nih.gov/pubmed/23192028. > > However I looked quite some time now, to find how to put the 100 ul 1% > low-melting agarose plug in the middle of the capillary, but was not > successful. Does one use a gel loading tip or is there a better way to do > this? > > Thanks in advance for any tips and tricks, > > best regards, Georg. >
Re: [ccp4bb] capillary counterdiffusion agarose plug
Hello Georg, You can melt the agarose in a glass container using a microwave oven. Then connect your capillary tube to small syringe, using a plastic tubing or adapter. Use some parafilm if the tubing diameters do not fit exactly, there should be no leaks. Then slowly pull the plunger to take ~100ul of agarose and keep pulling outside the liquid, until the plug is more or less in the center of the tube, leaving room for the precipitant and protein on each side. But most importantly, bear in mind that the system in the paper you cite works because the mother liquor components can diffuse readily through the agarose plug (MPD, NaAc, AmSO4, CaCl2). This might not be the case if your precipitant has high molecular weight PEG or the like, and your crystals need lower temperature to grow. Good luck, Javier On Mon, Jan 5, 2015 at 11:25 AM, Georg Mlynek wrote: > Dear Colleagues, > > After reading a few papers about growing suitable crystals for neutron > diffraction. I will do capillary counterdiffusion with an agarose plug > between mother liquor and protein solution like described in > http://www.ncbi.nlm.nih.gov/pubmed/23192028. > > However I looked quite some time now, to find how to put the 100 ul 1% > low-melting agarose plug in the middle of the capillary, but was not > successful. Does one use a gel loading tip or is there a better way to do > this? > > Thanks in advance for any tips and tricks, > > best regards, Georg. > -- Javier M. Gonzalez, PhD. Protein Crystallography Station Bioscience Division Bioenergy and Biome Sciences Group (B-11) Los Alamos National Laboratory TA-3, Building 4200, Room 202B Mailstop T007 Los Alamos, NM 87545 Phone: +1 (505) 667-9376 LinkedIn
Re: [ccp4bb] capillary counterdiffusion agarose plug
Dear Georg, There are different ways to set-up counterdiffusion experiment but the set-up you mentioned is done in a single capillary. The easiest way is to prepare the agarose and when it is still warm pipette the 100 microL on any surface, I use a piece of parafilm. Them you have to adsorb it with the help of a silicon-tube or syringe connected to one end of the capillary (see Fig. 1b of Acta Cryst. (2010). F66, 264–268). Place the small column of agarose at the center of the capillary and allow it gel. You can place the capillaries in the fridge to speed it up. When the agarose has gel you may add the protein and precipitant solution each one to one end of the capillaries using Hamilton syringes or capillaries of small diameter. Finally seal both ends with either bee-wax or vacuum grease. Cheers Gavi. 2015-01-05 18:25 GMT+01:00 Georg Mlynek : > Dear Colleagues, > > After reading a few papers about growing suitable crystals for neutron > diffraction. I will do capillary counterdiffusion with an agarose plug > between mother liquor and protein solution like described in > http://www.ncbi.nlm.nih.gov/pubmed/23192028. > > However I looked quite some time now, to find how to put the 100 ul 1% > low-melting agarose plug in the middle of the capillary, but was not > successful. Does one use a gel loading tip or is there a better way to do > this? > > Thanks in advance for any tips and tricks, > > best regards, Georg. > -- Dr. José A. Gavira Gallardo Laboratorio de Estudios Cristalográficos IACT, (CSIC-UGR) Av. de las Palmeras, 4 18100 Armilla (Granada) Tel.: 958 23 Ext.190205 Fax: 958 55 26 20 e-mail: jgav...@iact.ugr-csic.es web: http://www.lec.csic.es/gavi
Re: [ccp4bb] .raw file from SAINT to pointless
Dear George and Phil, thanks a lot for the fast answers. Things are unfortunately a bit more complicated and the usually very convenient way using SAINT-SADABS-XPREP has too much limitations for this datasets because 1. It starts with that one datasets has more than 2.000.000 reflections (space group P1, high redundancy and high resolution), so I already have to split the initial datasets in two. (SADABS can just process 2.000.000 reflections, probably something archaic from old days, when computers were not so fast?) 2. I can then of course combine them with xprep but the XPREP (version 2014/2) writes out just merged .sca file. Other formats hkl4, hkl3 are unmerged but can be just used with the command line version of xtriage and phenix merging statistics (which is a part of the phenix suite and needs additional inputs (spacegroup). However as aimless writes out all the statistics too, it is not necessary to run these phenix programs anyhow. Thank you both of you again, for the great programs and support. @Phil I will send you the SAINT manual and a small raw file offlist. @George can I write out unmerged .sca files from xprep and does it also print out CC*? (Of course I always use the latest ccp4, but I hoped the old one might work in my case).
[ccp4bb] Question on Refmac5
Dear All, When I run Refmac, it would produce a refined PDB file and mtz file. My question is, if I started the refinement at 6:00 pm and completed at 8:00 pm, I find the refined PDB file and mtz file were created in the target directory at perhaps 6:30 pm. I think from 6:30 pm the created PDB file and mtz file in the target directory would be automatically updated to 8: 00 pm when the refinement finished, am I right? Dialing
Re: [ccp4bb] Question on Refmac5
Dear Dialing are you talking about the 'creating date' or the 'modified date'? Leo > On Jan 6, 2015, at 2:55 AM, Dialing Pretty > <03f1d08ed29c-dmarc-requ...@jiscmail.ac.uk> wrote: > > Dear All, > > When I run Refmac, it would produce a refined PDB file and mtz file. My > question is, if I started the refinement at 6:00 pm and completed at 8:00 pm, > I find the refined PDB file and mtz file were created in the target directory > at perhaps 6:30 pm. I think from 6:30 pm the created PDB file and mtz file in > the target directory would be automatically updated to 8: 00 pm when the > refinement finished, am I right? > > Dialing
Re: [ccp4bb] Question on Refmac5
I am talking the "creating date". For my situation, once the PDB file and mtz file were created at around 6:00 pm, with the progression of the refinement and completed at 8:00 pm, the date shown in the directory folder is always 6:00 pm. After 8:00 pm when the refinement finished, I check the property of the PDB file and MTZ file, I find the modify time (should be by Refmac5) is only several seconds after the created time and the visited time, and the created time and the visited time are same. Clearly I cannot get the expected PDB file and the MTZ file after 2 hours refinement. Is any bug in my CCP4? Or there is something I do not understand? Dialing On Tuesday, 6 January 2015, 10:10, CHAVAS Leonard wrote: Dear Dialing are you talking about the 'creating date' or the 'modified date'? Leo On Jan 6, 2015, at 2:55 AM, Dialing Pretty <03f1d08ed29c-dmarc-requ...@jiscmail.ac.uk> wrote: Dear All, When I run Refmac, it would produce a refined PDB file and mtz file. My question is, if I started the refinement at 6:00 pm and completed at 8:00 pm, I find the refined PDB file and mtz file were created in the target directory at perhaps 6:30 pm. I think from 6:30 pm the created PDB file and mtz file in the target directory would be automatically updated to 8: 00 pm when the refinement finished, am I right? Dialing
