[ccp4bb] CCP4-6.4.0 Update 016
Dear CCP4 users, An update for the CCP4-6.4.0 series has just been released, consisting of the following changes: * prelyscar: New program, predictor of lysine caboxylation * CCP4i: prelyscar, new interface * pdb2to3: fixed for new ccp4srs * rapper: downgrade optimisation to workaround segv on OS X * Xia2: workaround aimless ASSERT * aimless: bug fixes, update to 0.3.5 * pdbse: copying chain ID into segment ID fields blocked for all PDB files except if marked for XPLOR use * cparrot: bug fixes Note that auto-updates work only with CCP4 6.4.0 series, therefore please upgrade if necessary. The Update Manager is now included in the package so you do not need to install it separately. In addition, all available updates will be installed automatically if you are using Setup Manager for CCP4 installation. Please report any bugs to c...@stfc.ac.uk. Many thanks for using CCP4. The CCP4 Core Team -- Scanned by iCritical.
[ccp4bb] Xia2 / XDS issues
Dear CCP4-ers. I am (trying) to use Xia2 (svn/Build 4599) to process some diffraction data. However, I am coming across the following issue, which I don’t seem to be able to solve... Integrating SWEEP1 Status: error [XDS] cannot allocate memory” I have checked XDS, and I have the correct (64-bit version installed: VERSION January 10, 2014 BUILT=20140307). I also have set the parameter KMP_STACKSIZE to 8m, in my .bash_profile as indicated in the XDS setup instructions. I am running OS X Mavericks (10.9.3) / XQuartz 2.7.6. I also have 16 GB of RAM installed. Any help / suggestions would be very much appreciated. Tony. - - - - - - - - - - - - - - - - - - Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ - - - - - - - - - - - - - - - - - - email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 http://www.sussex.ac.uk/lifesci/oliverlab http://tinyurl.com/aw-oliver - - - - - - - - - - - - - - - - - -
Re: [ccp4bb] Xia2 / XDS issues
Dear Antony, Have you try to use the option - parralel 1, to see what hapen. Maybe it's problem with the parallelism option. If you try this option you force to use only one core and i think you decrease your need in memory. If this solution help, you probably have to check the xdsInput parameters controled by xia2 if it's possible (i have not try at this time) . Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Antony Oliver [antony.oli...@sussex.ac.uk] Envoyé : lundi 19 mai 2014 14:37 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Xia2 / XDS issues Dear CCP4-ers. I am (trying) to use Xia2 (svn/Build 4599) to process some diffraction data. However, I am coming across the following issue, which I don’t seem to be able to solve... Integrating SWEEP1 Status: error [XDS] cannot allocate memory” I have checked XDS, and I have the correct (64-bit version installed: VERSION January 10, 2014 BUILT=20140307). I also have set the parameter KMP_STACKSIZE to 8m, in my .bash_profile as indicated in the XDS setup instructions. I am running OS X Mavericks (10.9.3) / XQuartz 2.7.6. I also have 16 GB of RAM installed. Any help / suggestions would be very much appreciated. Tony. - - - - - - - - - - - - - - - - - - Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ - - - - - - - - - - - - - - - - - - email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 http://www.sussex.ac.uk/lifesci/oliverlab http://tinyurl.com/aw-oliver - - - - - - - - - - - - - - - - - -
[ccp4bb] HisTrap Trap
Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem.). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often.see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved - the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious 'proprietary dispersant' preventing cell adhesion quoted.. Best wishes, BR Bernhard Rupp mailto:b...@ruppweb.org b...@ruppweb.org mailto:b...@hofkristallamt.org b...@hofkristallamt.org http://www.ruppweb.org/ http://www.ruppweb.org/ ---
Re: [ccp4bb] HisTrap Trap
Well, this is of only possible relevance, but in a previous lab, we used sf9 cells/media quite a bit, and there was always an issue similar to this, due to [we thought] ferritin being secreted into the medium, and sucking up the metals. Many, in fact, crystallized ferritin this way by mistake! Is the concentrated flow-through colored, indicating protein-bound metals, or do the metals go through a concentrator, indicating free or small-molecule-complexed metals? What about pH? What about Gibco's formulation-is it available? The mysterious dispersant could easily be either some chelator like EDTA. You could try ITC with Gibco in the cuvette, titrate with metals if you have a machine handy-it's just an hour or so. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Monday, May 19, 2014 10:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HisTrap Trap Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem...). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often...see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved - the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious 'proprietary dispersant' preventing cell adhesion quoted Best wishes, BR Bernhard Rupp b...@ruppweb.orgmailto:b...@ruppweb.org b...@hofkristallamt.orgmailto:b...@hofkristallamt.org http://www.ruppweb.org/ ---
Re: [ccp4bb] Xia2 / XDS issues
Dear Tony, Sorry to hear you are having trouble - I have never seen this message before. Please could you send me (off list) the output of the last XDS job ran before this one and (ideally) the XDS.INP file in the offending area? There must be something very odd going on here. Thanks best wishes, Graeme On 19 May 2014 13:37, Antony Oliver antony.oli...@sussex.ac.uk wrote: Dear CCP4-ers. I am (trying) to use Xia2 (svn/Build 4599) to process some diffraction data. However, I am coming across the following issue, which I don’t seem to be able to solve... Integrating SWEEP1 Status: error [XDS] cannot allocate memory” I have checked XDS, and I have the correct (64-bit version installed: VERSION January 10, 2014 BUILT=20140307). I also have set the parameter KMP_STACKSIZE to 8m, in my .bash_profile as indicated in the XDS setup instructions. I am running OS X Mavericks (10.9.3) / XQuartz 2.7.6. I also have 16 GB of RAM installed. Any help / suggestions would be very much appreciated. Tony. - - - - - - - - - - - - - - - - - - Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ - - - - - - - - - - - - - - - - - - email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 http://www.sussex.ac.uk/lifesci/oliverlab http://tinyurl.com/aw-oliver - - - - - - - - - - - - - - - - - -
Re: [ccp4bb] HisTrap Trap
Hi Bernhard, I just stumbled over this patent, where they add cobalt or nickel ions: http://www.google.com/patents/WO2013082518A1?cl=en [0086] Supplementing cell culture media, such as CD FortiCHO and Freestyle 293, with metal ions does prevent column stripping and improve histidine tagged protein binding during IMAC purification. This procedure works for multiple different IMAC columns including Ni-NTA, TALON® metal affinity, POROS® MC, as well as ProBond Ni-IDA (data not shown) resins. A variety of metals can be used including nickel, copper, and cobalt at concentrations ranging from 0.05 - 10 mM. MgCl2, however, did not appear to improve histidine tagged protein binding to IMAC columns. The optimal concentration for improving protein binding seems to be around 0.5mM for nickel and copper. The binding times tested varied from minutes (column protocol) to 4 hours (batch protocol) and in all cases supplementation with additional metal ions led to increased protein recoveries during IMAC purification, thus indicating the efficacy of this novel procedure. Wished I knew this years ago, cheers, Hans Bernhard Rupp schreef: Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem.). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often.see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved - the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious 'proprietary dispersant' preventing cell adhesion quoted.. Best wishes, BR Bernhard Rupp mailto:b...@ruppweb.org b...@ruppweb.org mailto:b...@hofkristallamt.org b...@hofkristallamt.org http://www.ruppweb.org/ http://www.ruppweb.org/ ---
Re: [ccp4bb] HisTrap Trap
I think the new versions of GE's HisTrap columns can address these problems. Try contacting someone at GE. I think a number of labs have had such problems in the past and the culprit has been believed to be a chelator in the media but never confirmed because the manufacturer of the media is not willing to disclose the contents for the media. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org Original message Date: Mon, 19 May 2014 14:38:12 + From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Keller, Jacob kell...@janelia.hhmi.org) Subject: Re: [ccp4bb] HisTrap Trap To: CCP4BB@JISCMAIL.AC.UK Well, this is of only possible relevance, but in a previous lab, we used sf9 cells/media quite a bit, and there was always an issue similar to this, due to [we thought] ferritin being secreted into the medium, and sucking up the metals. Many, in fact, crystallized ferritin this way by mistake! Is the concentrated flow-through colored, indicating protein-bound metals, or do the metals go through a concentrator, indicating free or small-molecule-complexed metals? What about pH? What about Gibco's formulation--is it available? The mysterious dispersant could easily be either some chelator like EDTA. You could try ITC with Gibco in the cuvette, titrate with metals if you have a machine handy--it's just an hour or so. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Monday, May 19, 2014 10:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HisTrap Trap Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem...). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often...see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved - the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious `proprietary dispersant' preventing cell adhesion quoted Best wishes, BR -- -- Bernhard Rupp b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ -- -
Re: [ccp4bb] HisTrap Trap
Bernhard, We have had similar, but not identical issues with some insect cell media, as well as column interference by lipid. If we see this, we tend to run all the material with the protein (cell lysate or media) through step-elution ion exchange (quick on, quick off). While the purification is minimal, the protein is more concentrated and performs better on Ni-columns. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 19, 2014, at 10:13 AM, Bernhard Rupp hofkristall...@gmail.com wrote: Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem…). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often…see yield remark). Overcome by common crystallographers’ greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved – the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious ‘proprietary dispersant’ preventing cell adhesion quoted…. Best wishes, BR Bernhard Rupp b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ ---
Re: [ccp4bb] HisTrap Trap
If EDTA is the issue (as a secret component) then I can see that Mg does not help, because the Binding(Chelating) constant is 10 orders of magnitude higher for Ni than Mg. Ni or Co could however work to saturate the EDTA. pH (too basic) I don't think so because the cells would not react well to any significant change. Color changes I cannot see, largely because the loaded medium is orange anyhow. Thanks for the responses, more testingright now: good old fashioned dialysis into the HisTrap loading buffer Thx, BR -Original Message- From: H. Raaijmakers [mailto:hraaijmak...@xs4all.nl] Sent: Montag, 19. Mai 2014 16:55 To: b...@hofkristallamt.org Cc: ccp4bb@jiscmail.ac.uk Subject: Re: [ccp4bb] HisTrap Trap Hi Bernhard, I just stumbled over this patent, where they add cobalt or nickel ions: http://www.google.com/patents/WO2013082518A1?cl=en [0086] Supplementing cell culture media, such as CD FortiCHOT and Freestyle 293, with metal ions does prevent column stripping and improve histidine tagged protein binding during IMAC purification. This procedure works for multiple different IMAC columns including Ni-NTA, TALONR metal affinity, POROSR MC, as well as ProBondT Ni-IDA (data not shown) resins. A variety of metals can be used including nickel, copper, and cobalt at concentrations ranging from 0.05 - 10 mM. MgCl2, however, did not appear to improve histidine tagged protein binding to IMAC columns. The optimal concentration for improving protein binding seems to be around 0.5mM for nickel and copper. The binding times tested varied from minutes (column protocol) to 4 hours (batch protocol) and in all cases supplementation with additional metal ions led to increased protein recoveries during IMAC purification, thus indicating the efficacy of this novel procedure. Wished I knew this years ago, cheers, Hans Bernhard Rupp schreef: Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem.). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often.see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved - the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious 'proprietary dispersant' preventing cell adhesion quoted.. Best wishes, BR -- -- Bernhard Rupp mailto:b...@ruppweb.org b...@ruppweb.org mailto:b...@hofkristallamt.org b...@hofkristallamt.org http://www.ruppweb.org/ http://www.ruppweb.org/ -- -
Re: [ccp4bb] HisTrap Trap
Bernhard, One other point, after rereading your email. For step-elution ion exchange (quick on, quick off), you can use some very cheap and quite robust media the will resist a lot of stuff. Have them pour their own columns and toss it away when it gets too dirty. You don't have to use the high-end stuff for this step (CM-sephedex, Dowex, CM-cellulose, etc.). Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 19, 2014, at 10:13 AM, Bernhard Rupp hofkristall...@gmail.com wrote: Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem…). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often…see yield remark). Overcome by common crystallographers’ greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved – the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious ‘proprietary dispersant’ preventing cell adhesion quoted…. Best wishes, BR Bernhard Rupp b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ ---
Re: [ccp4bb] HisTrap Trap
I doubt it is ferritin sucking up metals; although the problem of crystallizing ferritin is an issue in secreted preps. See this paper, where they made lemonade (a PNAS paper) from this lemon (High Five ferritin crystals): Secreted ferritin was isolated from the supernatants of baculovirus-infected T. ni cells as a contaminant during purification of an unrelated recombinant 6xHis-tagged protein on a Ni-agarose column... http://www.ncbi.nlm.nih.gov/pubmed/15896348 The reason why I don't think it is ferritin is if you buffer exchange your entire secreted prep, you still end up with ferritin contaminating your prep, but your resin is not ruined. Overall, methods that don't remove any protein, but only small-molecule components perform perfectly well preserving resin in secreted insect/mammalian preps. So, your second guess is probably correct. By the way, I have once quizzed the makers of one proprietary medium, and their response was Yes, IMAC won't work, and no, we cannot tell you what it is that makes it not work, it is proprietary. Best, Engin On 5/19/14, 9:38 AM, Keller, Jacob wrote: Well, this is of only possible relevance, but in a previous lab, we used sf9 cells/media quite a bit, and there was always an issue similar to this, due to [we thought] ferritin being secreted into the medium, and sucking up the metals. Many, in fact, crystallized ferritin this way by mistake! Is the concentrated flow-through colored, indicating protein-bound metals, or do the metals go through a concentrator, indicating free or small-molecule-complexed metals? What about pH? What about Gibco's formulation---is it available? The mysterious dispersant could easily be either some chelator like EDTA. You could try ITC with Gibco in the cuvette, titrate with metals if you have a machine handy---it's just an hour or so. JPK *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Bernhard Rupp *Sent:* Monday, May 19, 2014 10:14 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] HisTrap Trap Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem...). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often...see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved -- the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious 'proprietary dispersant' preventing cell adhesion quoted Best wishes, BR Bernhard Rupp b...@ruppweb.org mailto:b...@ruppweb.org b...@hofkristallamt.org mailto:b...@hofkristallamt.org http://www.ruppweb.org/ ---
Re: [ccp4bb] HisTrap Trap
I use already the HisTrap Excel - they really do not leach under normal treatment. Chelator is a prime suspect, agreed. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Reza Khayat Sent: Montag, 19. Mai 2014 17:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] HisTrap Trap I think the new versions of GE's HisTrap columns can address these problems. Try contacting someone at GE. I think a number of labs have had such problems in the past and the culprit has been believed to be a chelator in the media but never confirmed because the manufacturer of the media is not willing to disclose the contents for the media. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org Original message Date: Mon, 19 May 2014 14:38:12 + From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Keller, Jacob kell...@janelia.hhmi.org) Subject: Re: [ccp4bb] HisTrap Trap To: CCP4BB@JISCMAIL.AC.UK Well, this is of only possible relevance, but in a previous lab, we used sf9 cells/media quite a bit, and there was always an issue similar to this, due to [we thought] ferritin being secreted into the medium, and sucking up the metals. Many, in fact, crystallized ferritin this way by mistake! Is the concentrated flow-through colored, indicating protein-bound metals, or do the metals go through a concentrator, indicating free or small-molecule-complexed metals? What about pH? What about Gibco's formulation--is it available? The mysterious dispersant could easily be either some chelator like EDTA. You could try ITC with Gibco in the cuvette, titrate with metals if you have a machine handy--it's just an hour or so. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Monday, May 19, 2014 10:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HisTrap Trap Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem...). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often...see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved - the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious `proprietary dispersant' preventing cell adhesion quoted Best wishes, BR -- -- Bernhard Rupp b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ -- -
Re: [ccp4bb] HisTrap Trap
Our HEK medium has some pH-indicator dye in it, and phenol red (phenolsulfonphtalein) is a good guess. Under the assumption that the same indicator was in the other CHO medium, it did not cause a problem there. Thx! BR -Original Message- From: Oganesyan, Vaheh [mailto:oganesy...@medimmune.com] Sent: Montag, 19. Mai 2014 17:38 To: b...@hofkristallamt.org Subject: RE: [ccp4bb] HisTrap Trap Orange to pink colored media is a sign of phenol presence which is added to buffer. That phenol is the problem. I was having similar problem and asked not to add it. Everything worked well afterwards. Vaheh Oganesyan www.medimmune.com -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Monday, May 19, 2014 11:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] HisTrap Trap If EDTA is the issue (as a secret component) then I can see that Mg does not help, because the Binding(Chelating) constant is 10 orders of magnitude higher for Ni than Mg. Ni or Co could however work to saturate the EDTA. pH (too basic) I don't think so because the cells would not react well to any significant change. Color changes I cannot see, largely because the loaded medium is orange anyhow. Thanks for the responses, more testingright now: good old fashioned dialysis into the HisTrap loading buffer Thx, BR -Original Message- From: H. Raaijmakers [mailto:hraaijmak...@xs4all.nl] Sent: Montag, 19. Mai 2014 16:55 To: b...@hofkristallamt.org Cc: ccp4bb@jiscmail.ac.uk Subject: Re: [ccp4bb] HisTrap Trap Hi Bernhard, I just stumbled over this patent, where they add cobalt or nickel ions: http://www.google.com/patents/WO2013082518A1?cl=en [0086] Supplementing cell culture media, such as CD FortiCHOT and Freestyle 293, with metal ions does prevent column stripping and improve histidine tagged protein binding during IMAC purification. This procedure works for multiple different IMAC columns including Ni-NTA, TALONR metal affinity, POROSR MC, as well as ProBondT Ni-IDA (data not shown) resins. A variety of metals can be used including nickel, copper, and cobalt at concentrations ranging from 0.05 - 10 mM. MgCl2, however, did not appear to improve histidine tagged protein binding to IMAC columns. The optimal concentration for improving protein binding seems to be around 0.5mM for nickel and copper. The binding times tested varied from minutes (column protocol) to 4 hours (batch protocol) and in all cases supplementation with additional metal ions led to increased protein recoveries during IMAC purification, thus indicating the efficacy of this novel procedure. Wished I knew this years ago, cheers, Hans Bernhard Rupp schreef: Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem.). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often.see yield remark). Overcome by common crystallographers' greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved - the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious 'proprietary dispersant' preventing cell adhesion quoted.. Best wishes, BR -- -- Bernhard Rupp mailto:b...@ruppweb.org b...@ruppweb.org mailto:b...@hofkristallamt.org b...@hofkristallamt.org http://www.ruppweb.org/ http://www.ruppweb.org/ -- - To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation. To the extent this electronic communication or any of its attachments contain information that is
[ccp4bb] data processing with XDS
Dear all, I am looking for some suggestions here. I have lately collected some datasets but the spots are very very weak... it is very difficult to process them. At times it looks like XDS is stalled or at times it just says that it can not interpret the lattice parameters... also while running integrate I have such a message after each range of images: !!! WARNING !!! REFINEMENT DID NOT CONVERGE LAST CORRECTION SHIFT WAS 9.1E-03 (should be 1.0E-03) I guess this is not good at all. And I wonder what to do? Is there any info that I can get out from these data at all? or rather not? I wonder if the problem is to find the spots, I have tried with HKL2000 and it can't even find enough of them for indexing. Any ideas are welcome. Best wishes, Almudena. -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] Xia2 / XDS issues
Dear CCP4-ers. Many apologies for the previous post (with attachments). However, it is true - somewhat crappy data, that I can’t seem to reprocess with XDS / xia2. Tony. HI Graeme, Please find below, what I think it is that you need? Files attached, plus relevant text clips,. NB: everything seems to fail at the integration step. NB2: This is quite crappy data….(!) Tony. - - - - - - - - - - - - - - - - - - Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ - - - - - - - - - - - - - - - - - - email: antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 http://www.sussex.ac.uk/lifesci/oliverlab http://tinyurl.com/aw-oliver - - - - - - - - - - - - - - - - - -
Re: [ccp4bb] Xia2 / XDS issues
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Antony, you should check images 189, 190,... XDS only finds 2 strong reflections (NSTRONG). As the LP-file indicates, these are not enough to carry out refinement. My guess is these are blank images - XDS can skip over them if they are not too many, but not always. I would not be surprised if the error message goes away once you fixed this problem. It may be some stray effect from the finding not enough reflections. If you do insist on processing these images, swich off refinement REFINE(INTEGRATE)=! in XDS.INP Best, Tim On 05/19/2014 06:09 PM, Antony Oliver wrote: HI Graeme, Please find below, what I think it is that you need? Files attached, plus relevant text clips,. NB: everything seems to fail at the integration step. NB2: This is quite crappy data….(!) Tony. XDS.INP JOB=INTEGRATE MAXIMUM_NUMBER_OF_PROCESSORS=8 TEST=2 DELPHI=5.0 REFINE(INTEGRATE)=ORIENTATION CELL BEAM DISTANCE DETECTOR=ADSC MINIMUM_VALID_PIXEL_VALUE=1 OVERLOAD=65000 DIRECTION_OF_DETECTOR_X-AXIS=1.00 0.00 0.00 DIRECTION_OF_DETECTOR_Y-AXIS=0.00 1.00 -0.00 TRUSTED_REGION=0.0 1.41 NX=3072 NY=3072 QX=0.102600 QY=0.102600 DETECTOR_DISTANCE=456.750 OSCILLATION_RANGE=2.00 X-RAY_WAVELENGTH=0.979500 ROTATION_AXIS= 1.000 0.000 0.000 INCIDENT_BEAM_DIRECTION=0.0 0.0 1.0 FRACTION_OF_POLARIZATION=0.99 POLARIZATION_PLANE_NORMAL=0.0 1.0 0.0 AIR=0.001 NAME_TEMPLATE_OF_DATA_FRAMES=/Volumes/Data/diamond/I02_0211/Mohan/TNK1/xtal1/xtal1_M1S1_4_???.img DATA_RANGE=1 200 9_xds_par_INTEGRATE.log ** PROCESSING OF IMAGES 189 ... 191 ** USING 3 PROCESSORS *** DEFINITION OF SYMBOLS *** IER = ERROR CODE AFTER ACCESSING DATA IMAGE 0: NO ERROR -1: CANNOT OPEN OR READ IMAGE FILE -3: WRONG DATA FORMAT SCALE = SCALING FACTOR FOR THIS DATA IMAGE NBKG= NUMBER OF BACKGROUND PIXELS ON DATA IMAGE NOVL= NUMBER OF OVERLOADED REFLECTIONS ON DATA IMAGE NEWALD = NUMBER OF REFLECTIONS CLOSE TO THE EWALD SPHERE NSTRONG = NUMBER OF STRONG REFLECTIONS ON DATA IMAGE NREJ= NUMBER OF UNEXPECTED REFLECTIONS SIGMAB = BEAM_DIVERGENCE_E.S.D.=SIGMAB SIGMAR = REFLECTING_RANGE_E.S.D.=SIGMAR (MOSAICITY) IMAGE IER SCALE NBKG NOVL NEWALD NSTRONG NREJ SIGMAB SIGMAR 189 0 1.038 90884180 14057 2 0 0.0069 3. 190 0 1.037 90870920 14088 0 0 0. 0. 191 0 1.039 90879820 14075 2 0 0.0260 0.9701 !!! WARNING !!! UNABLE TO CARRY OUT REFINEMENT. RETURN CODE IER= 0 XDS WILL CONTINUE WITH ORIGINAL DIFFRACTION PARAMETER VALUES. !!! ERROR !!! CANNOT ALLOCATE MEMORY YOU COULD RERUN THIS STEP WITH SMALLER VALUES FOR THE PARAMETERS NUMBER_OF_PROFILE_GRID_POINTS_ALONG_ALPHA/BETA= NUMBER_OF_PROFILE_GRID_POINTS_ALONG_GAMMA= # command line: # xds_par - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTejIvUxlJ7aRr7hoRAhxKAKDXguJTekWZg+0HZtQF1VvaOdreFgCdG/68 lEeZGkltEMVsbZoL7/UKhvE= =xyOf -END PGP SIGNATURE-
Re: [ccp4bb] HisTrap Trap
I worked for several years on mammalian cell expression of extra-cellular domains of single spanning membrane proteins and other membrane proteins. Just like you did when we switched stable lines from serum containing to serum free medium the expression drastically reduced. So after switching to serum free medium did you try picking clones in selective medium again and try to look for adapted clones to the new medium? No protocol suggest this but i had good luck. I am not very sure this is exactly your problem if so what you see is not very unusual. I do not understadn why you switch from your CHO stable line expression? In my opinion stick with stable clones in serum containing medium or serum free medium (not really necessary since it will run very expensive in the long run) since you are using affinity tags to pull your protein of interest from secreted protein in the medium a batch binding of your media in a roller bottle apparatus over several hours will help solve your binding issues. Identify ideal washes to get rid of serum binding or reduce non-specific binding. I am surprised how hyper-glycosylation was not a problem when HEK is used. To save a bunch of money on deglycosylation enzymes you should switch to LEC mutants of CHO cell lines. Padayatti On Mon, May 19, 2014 at 7:13 AM, Bernhard Rupp hofkristall...@gmail.comwrote: Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem…). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often…see yield remark). Overcome by common crystallographers’ greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved – the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious ‘proprietary dispersant’ preventing cell adhesion quoted…. Best wishes, BR Bernhard Rupp b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ --- -- P
Re: [ccp4bb] data processing with XDS
Hi Almudena Have you tried Mosflm (since this is the CCP4 BB...)? On 19 May 2014, at 17:18, Almudena Ponce Salvatierra wrote: Dear all, I am looking for some suggestions here. I have lately collected some datasets but the spots are very very weak... it is very difficult to process them. At times it looks like XDS is stalled or at times it just says that it can not interpret the lattice parameters... also while running integrate I have such a message after each range of images: !!! WARNING !!! REFINEMENT DID NOT CONVERGE LAST CORRECTION SHIFT WAS 9.1E-03 (should be 1.0E-03) I guess this is not good at all. And I wonder what to do? Is there any info that I can get out from these data at all? or rather not? I wonder if the problem is to find the spots, I have tried with HKL2000 and it can't even find enough of them for indexing. Any ideas are welcome. Best wishes, Almudena. -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
Re: [ccp4bb] data processing with XDS
Hi Almudena, you can also try mosflm as said Harry. And you can try different setting in the XDS.INP, you can try to reduce the STRONG_PIXEL (because you said spots are weak) number or/and MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT (if the spot are small). Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Almudena Ponce Salvatierra [maps.fa...@gmail.com] Envoyé : lundi 19 mai 2014 18:18 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] data processing with XDS Dear all, I am looking for some suggestions here. I have lately collected some datasets but the spots are very very weak... it is very difficult to process them. At times it looks like XDS is stalled or at times it just says that it can not interpret the lattice parameters... also while running integrate I have such a message after each range of images: !!! WARNING !!! REFINEMENT DID NOT CONVERGE LAST CORRECTION SHIFT WAS 9.1E-03 (should be 1.0E-03) I guess this is not good at all. And I wonder what to do? Is there any info that I can get out from these data at all? or rather not? I wonder if the problem is to find the spots, I have tried with HKL2000 and it can't even find enough of them for indexing. Any ideas are welcome. Best wishes, Almudena. -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] metal chelation
Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
Are you treating all your buffers with metal chelating resin? Are you washing all plasticware with EDTA and metal-free buffer prior to use? How are you quantifying your metal content, and what metal ions are contaminating your sample? You might be pulling out the metal ions, but they get right back in as soon as you remove the chelators. Metal ions are present at trace levels in all water and chemicals (Zn is particularly common). You might need to 'soften up' the protein a little bit, we use a pH 3.8 stripping protocol. Denaturants may be required. Time is also a factor, extending your chelating step might help. It's not trivial to make app-enzymes, in my experience, Nat On Mon, May 19, 2014 at 12:20 PM, SUBSCRIBE CCP4BB Adam Brummett adam-brumm...@uiowa.edu wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Adam, - - many plasmids for His-tags contain a cleavage site to remove the tag. In fact this provides you with an additional purifiction step which is complementary to the first Ni-column and therefore quite a good strategy (in addition to chopping off the chelating His-tag). - - you can try other Titriplexes like EGTA, EDTA is only one of a series - - if only the His-tag binds the ion, wouldn't this qualify the protein as 'apo'? If the His-tag is ordered, you could actually replace the ion with Co and use this for phasing, in case this is an issue. Best, Tim On 05/19/2014 07:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTekO/UxlJ7aRr7hoRAkhUAKCXbpCx2x4arUbTDtnpDdviIoL3/QCeP+ik aaDa+rmp1AALJhdsDn8els0= =hfzc -END PGP SIGNATURE-
Re: [ccp4bb] HisTrap Trap
Hi Bernhard, I too suspect that it is some kind of metal chelating reagent causing the problem (possibly used in medium for carrying Fe2+, as the free Fe2+ is toxic to cells). One simple test would be to load a liter of the unused medium to the Ni2+ column and see what happens. Do you concentrate your medium before pull-down? If it is the chelating reagent in the medium, then concentrating the medium 10-30x may help a lot (also helps yield, since every affinity binding has a Kd). We do that regularly on tangential flow filter columns. It is a little weird that your first run was OK but the later ones suffered from Ni2+ loss. I wonder if you can try stripping the column with EDTA first and then loading it again with fresh NiCl2, every time before loading the medium. The reason to strip it is that I also worry that some Ni2+ on your column might have been partially replaced by some metal ions from the medium. (Loading 1L medium to a 1mL column does not sound like a great idea to me anyways...) Zhijie From: Bernhard Rupp Sent: Monday, May 19, 2014 10:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HisTrap Trap Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem…). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often…see yield remark). Overcome by common crystallographers’ greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved – the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious ‘proprietary dispersant’ preventing cell adhesion quoted…. Best wishes, BR Bernhard Rupp b...@ruppweb.org b...@hofkristallamt.org http://www.ruppweb.org/ ---
Re: [ccp4bb] metal chelation
The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are not going to give up their metal to chelators, or just any chelator, or at all. Denaturation, dialysis, and refolding is an extreme way of removing metal ions to make apoprotein. Won't work for every protein. Chelation can be highly specific, that is one chelator may work, while another, similar one, will not. Some metal ions are notoriously difficult to eliminate, because they are adventitious trace contaminants in nearly everything, e.g. zinc and maybe even iron. (Plastic-ware seems to be often loaded with trace iron, and also is capable of adsorbing metal ions form solution.) To make apo-enzymes from zinc proteins, you have to go to heroic efforts to ensure that glassware, water, buffers, and reagents are zinc-free, especially if you don't have high (mM) concentrations of protein to work with. A His-tag is very likely to snag adventitious metals from solution, and can often mess up metal analysis for metalloproteins by providing extra metal. If this is a problem for your application, you may want to consider removing the His-tag. If you are making apoenzyme to get a different metal installed (metallosubstitution), there are slightly easier ways to do that than going through the apoenzyme route. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett rrowl...@colgate.edu wrote: The answer depends on a number of questions: What metal ion are you trying to eliminate? What kind of metal-binding site is involved? A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are not going to give up their metal to chelators, or just any chelator, or at all. Denaturation, dialysis, and refolding is an extreme way of removing metal ions to make apoprotein. Won't work for every protein. Chelation can be highly specific, that is one chelator may work, while another, similar one, will not. Some metal ions are notoriously difficult to eliminate, because they are adventitious trace contaminants in nearly everything, e.g. zinc and maybe even iron. (Plastic-ware seems to be often loaded with trace iron, and also is capable of adsorbing metal ions form solution.) To make apo-enzymes from zinc proteins, you have to go to heroic efforts to ensure that glassware, water, buffers, and reagents are zinc-free, especially if you don't have high (mM) concentrations of protein to work with. A His-tag is very likely to snag adventitious metals from solution, and can often mess up metal analysis for metalloproteins by providing extra metal. If this is a problem for your application, you may want to consider removing the His-tag. If you are making apoenzyme to get a different metal installed (metallosubstitution), there are slightly easier ways to do that than going through the apoenzyme route. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] HisTrap Trap
Hi Bernhard - Like Zhijie, we have also been using a Tangential Flow Filtration (TFF) system to address this same issue with serum-free 293 Freestyle media. Ours is the Minimate TFF system from Pall; there is also the Millipore Labscale system, and I’m sure others as well. I’ve done only limited direct testing, but I have seen 2- to 3-fold increased yield from a TFF’ed sample in comparison to a non-TFF'ed sample from the same batch of media. I concentrate the media 10x, then change the buffer by continuous diafiltration with 5-6 volumes of Ni-NTA loading/wash buffer. The advantages of TFF over plain dialysis are that it’s faster and uses less dialysis buffer, and due to the continuous agitation, the membrane is less likely to become clogged. On the downside, the membrane capsules are rather pricey, and although it’s mostly hands-off time, the process can still take quite a while, especially with lower-MWCO membranes. Good luck! Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center http://kong.med.nyu.edu/ On May 19, 2014, at 1:54 PM, Zhijie Li zhijie...@utoronto.camailto:zhijie...@utoronto.ca wrote: Hi Bernhard, I too suspect that it is some kind of metal chelating reagent causing the problem (possibly used in medium for carrying Fe2+, as the free Fe2+ is toxic to cells). One simple test would be to load a liter of the unused medium to the Ni2+ column and see what happens. Do you concentrate your medium before pull-down? If it is the chelating reagent in the medium, then concentrating the medium 10-30x may help a lot (also helps yield, since every affinity binding has a Kd). We do that regularly on tangential flow filter columns. It is a little weird that your first run was OK but the later ones suffered from Ni2+ loss. I wonder if you can try stripping the column with EDTA first and then loading it again with fresh NiCl2, every time before loading the medium. The reason to strip it is that I also worry that some Ni2+ on your column might have been partially replaced by some metal ions from the medium. (Loading 1L medium to a 1mL column does not sound like a great idea to me anyways...) Zhijie From: Bernhard Ruppmailto:hofkristall...@gmail.com Sent: Monday, May 19, 2014 10:13 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] HisTrap Trap Hi Fellows, my lab mates successfully expressed a glycoprotein in CHO cells in serum free medium, and the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield is not my problem…). We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole gradient. 20mM Imidazole buffer for regeneration. Works fine (and often…see yield remark). Overcome by common crystallographers’ greed (nor creed), we switched to stable xfected HEK293, and cell free medium Gibco CD 293. The first run gave high final yields cheers. The second run less of either, because the small HisTrap column essentially dissolved – the medium collapsed, Ni leaches out, kaput as kaput goes. A 3rd run on a similar previously working column lead to the same result. Only thing changed was the cells and medium. Same buffers, same gradients, same Akta equipment, same lab techs. Before I improve the statistics by ruining further columns, has anybody experienced such a calamity that might be blamable on secret media components or similar? There is a mysterious ‘proprietary dispersant’ preventing cell adhesion quoted…. Best wishes, BR Bernhard Rupp b...@ruppweb.orgmailto:b...@ruppweb.org b...@hofkristallamt.orgmailto:b...@hofkristallamt.org http://www.ruppweb.org/ --- This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. =
Re: [ccp4bb] metal chelation
Hi Adam, I have not read all the thread as it came all at once and late (9:00pm here). I believe the best way to strip a protein of metals is to first adsorb it onto a solid support (e.g. IEX) and then use a sufficiently low-pH (say equal or below 6) buffer that contains also EDTA. You will probably need several washes but it works! Also be aware that EDTA binds well to several proteins. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet at univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 19/05/2014 20:21, Adam Brummett wrote: Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu wrote: The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are not going to give up their metal to chelators, or just any chelator, or at all. Denaturation, dialysis, and refolding is an extreme way of removing metal ions to make apoprotein. Won't work for every protein. Chelation can be highly specific, that is one chelator may work, while another, similar one, will not. Some metal ions are notoriously difficult to eliminate, because they are adventitious trace contaminants in nearly everything, e.g. zinc and maybe even iron. (Plastic-ware seems to be often loaded with trace iron, and also is capable of adsorbing metal ions form solution.) To make apo-enzymes from zinc proteins, you have to go to heroic efforts to ensure that glassware, water, buffers, and reagents are zinc-free, especially if you don't have high (mM) concentrations of protein to work with. A His-tag is very likely to snag adventitious metals from solution, and can often mess up metal analysis for metalloproteins by providing extra metal. If this is a problem for your application, you may want to consider removing the His-tag. If you are making apoenzyme to get a different metal installed (metallosubstitution), there are slightly easier ways to do that than going through the apoenzyme route. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] metal chelation
Adam, We developed a protocol (loosely based on a few previous literature reports) for metallo-substitution of beta-carbonic anhydrase (a zinc-metalloenzyme) with cobalt(II). The metal ion in this enzyme is extremely refractory toward extraction with chelators, and the protein will not denature/refold at all. Briefly, our protocol involves overexpressing protein in minimal media in the presence of 10-100 uM Co(II) ion. This allows us nearly 100% metallosubstitution of Co(II) for Zn(II) during the overexpression phase. We have verified that our commercially prepared minimal media is quite metal-free. The protocol is described here: * Katherine M. Hoffmann,† Dejan Samardzic,* Katherine van den Heever,* and Roger S. Rowlett§, “Co(II)-substituted Haemophilus influenzae β-Carbonic Anhydrase: Spectral Evidence for Allosteric Regulation by pH and Bicarbonate Ion,” Arch. Biochem. Biophys. 2011, 511, 80-87. A couple of tricks we discovered: * Thiamin supplementation is required for good growth in minimal media * We have to use conditioned plastic expression flasks for this to work well. Acid-washed flasks result in no cell growth. Flasks that have been used to do regular overexpression runs, but have been simply well rinsed out with deionized water work reproducibly well. I'm pretty sure that the walls of the conditioned flasks are providing sufficient trace metal ions for growth, without swamping out our supplemental Co(II) ion with contaminant ions. FWIW, every time we do ICP analysis of metalloenzymes that are His-tagged, we nearly always get non-stoichiometric extra metal ion. It's maddening when you are trying to establish protein:metal stoichiometry, or determine accurate protein concentrations this way. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 3:04 PM, Nadir T. Mrabet wrote: Hi Adam, I have not read all the thread as it came all at once and late (9:00pm here). I believe the best way to strip a protein of metals is to first adsorb it onto a solid support (e.g. IEX) and then use a sufficiently low-pH (say equal or below 6) buffer that contains also EDTA. You will probably need several washes but it works! Also be aware that EDTA binds well to several proteins. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet at univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 19/05/2014 20:21, Adam Brummett wrote: Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett rrowl...@colgate.edu mailto:rrowl...@colgate.edu wrote: The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are
Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Hello all, Thank you for you suggestions. I took a look at the crystal packing for the solution with one molecule per asu, and the next closest molecule is 50 angstroms away, suggesting that this is not likely the correct solution. I have also tried MR with a number of different molecules per asu. In some cases I get better packing, but I have not yet gotten a solution that looks to refine well. In addition to my previous information, I would like to add the following: 1. The resolution of my data is not particular great. I get ~4 A resolution at best and spots are rather weak even with almost no beam attenuation. 2. The search model that gives the best solution (in terms of contrast score in MolRep) is an NMR structure. I have heard that MR with NMR structures can possibly give false solutions. An alternate crystal structure that I tried using gave much poorer contrast socres overall, regardless of the number of molecules in the asu to search for. If anyone has any additonal suggestion, I would appreciate them. Thanks, Matt On Fri, May 16, 2014 at 8:46 AM, R. M. Garavito rmgarav...@gmail.comwrote: Matt, In addition to the suggestions of the others, have you done a simple self rotation function? It can tell you quite a bit about how things are packed and give you strict criteria for choosing one solution over another. As Roger said, choosing an even number of monomers in the ASU is a good strategy, particularly if the self rotation function shows NCS 2-folds. Also, a calculated Matthews coefficient is NEVER correct, it is a mere guideline; it only has validity for any particularly crystal form AFTER the fact. Let the number of monomers in the ASU vary from 6-10; I have had MR cases that have had as little as 40% solvent to 70% solvent, where the calculated Matthews coefficient was quite wrong (i.e., the most common value observed in OTHER crystals). Two things to watch out for are: (1) An odd number of monomers in the ASU. I have had 1 1/2 dimers in an ASU (the 1/2 dimer is paired with another in a neighboring ASU). It is sometimes confusing to people and occasionally difficult to solve with some MR programs due to clashes. (2) Translation symmetry, which still can confuse some programs (but they are are getting better at detecting it). Finally, as Herman pointed out, look at the packing of any solution you are considering. It is surprising how a correct solution looks correct: nice intermolecular contacts and a pleasing distribution of mass throughout the unit cell (meaning expand out to at least a unit cell volume, which is easy in Pymol). Any unexplained gaps (meaning not caused by a missing domain) should be viewed critically. Regards and Good Luck, Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry Molecular Biology* *603 Wilson Rd., Rm. 513* *Michigan State University * *East Lansing, MI 48824-1319* *Office:* *(517) 355-9724 %28517%29%20355-9724 Lab: (517) 353-9125 %28517%29%20353-9125* *FAX: (517) 353-9334 %28517%29%20353-9334 Email: rmgarav...@gmail.com garav...@gmail.com* ** On May 15, 2014, at 6:50 PM, Matthew Bratkowski mab...@cornell.edu wrote: Hello all, I am working on the structure of a small protein in space group P212121. The protein is monomeric in solution based on gel filtration analysis. The Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit results in ~50% solvent content, while 1 molecule per asymmetric unit results in ~95% solvent. I tried molecular replacement with a search model which is essentially identical in sequence to my protein, and searched for 9 or 10 molecules/asu. Using MolRep with 9 or 10 molecules/asu, I get poor contrast scores around 1-1.5. However, when using Phaser, I get a solution with one molecules/asu. Likewise, when I went back and tried MolRep with 1 molecule/asu, I got a contrast score of 3.12. This model still has some issues, but looks more correct compaired to models created with 9 or 10 molecules/asu. It seems highly unlikely that a crystal would contain 95% solvent, but is there any possiblility that this could be the case? Assuming that the Matthews coefficient is correct, does anyone have an idea why MR seems to work better for 1 molecule/asu with 95% solvent content compared to 9-10 molecules with 50% solvent content? Alternatively, is there any reason why the Matthews coefficient could be calculating incorrectly? Any suggestions would be helpful. Thanks, Matt
Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement
Did you look at the maps for extra density/molecules? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Matthew Bratkowski Sent: Monday, May 19, 2014 4:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement Hello all, Thank you for you suggestions. I took a look at the crystal packing for the solution with one molecule per asu, and the next closest molecule is 50 angstroms away, suggesting that this is not likely the correct solution. I have also tried MR with a number of different molecules per asu. In some cases I get better packing, but I have not yet gotten a solution that looks to refine well. In addition to my previous information, I would like to add the following: 1. The resolution of my data is not particular great. I get ~4 A resolution at best and spots are rather weak even with almost no beam attenuation. 2. The search model that gives the best solution (in terms of contrast score in MolRep) is an NMR structure. I have heard that MR with NMR structures can possibly give false solutions. An alternate crystal structure that I tried using gave much poorer contrast socres overall, regardless of the number of molecules in the asu to search for. If anyone has any additonal suggestion, I would appreciate them. Thanks, Matt On Fri, May 16, 2014 at 8:46 AM, R. M. Garavito rmgarav...@gmail.commailto:rmgarav...@gmail.com wrote: Matt, In addition to the suggestions of the others, have you done a simple self rotation function? It can tell you quite a bit about how things are packed and give you strict criteria for choosing one solution over another. As Roger said, choosing an even number of monomers in the ASU is a good strategy, particularly if the self rotation function shows NCS 2-folds. Also, a calculated Matthews coefficient is NEVER correct, it is a mere guideline; it only has validity for any particularly crystal form AFTER the fact. Let the number of monomers in the ASU vary from 6-10; I have had MR cases that have had as little as 40% solvent to 70% solvent, where the calculated Matthews coefficient was quite wrong (i.e., the most common value observed in OTHER crystals). Two things to watch out for are: (1) An odd number of monomers in the ASU. I have had 1 1/2 dimers in an ASU (the 1/2 dimer is paired with another in a neighboring ASU). It is sometimes confusing to people and occasionally difficult to solve with some MR programs due to clashes. (2) Translation symmetry, which still can confuse some programs (but they are are getting better at detecting it). Finally, as Herman pointed out, look at the packing of any solution you are considering. It is surprising how a correct solution looks correct: nice intermolecular contacts and a pleasing distribution of mass throughout the unit cell (meaning expand out to at least a unit cell volume, which is easy in Pymol). Any unexplained gaps (meaning not caused by a missing domain) should be viewed critically. Regards and Good Luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724tel:%28517%29%20355-9724 Lab: (517) 353-9125tel:%28517%29%20353-9125 FAX: (517) 353-9334tel:%28517%29%20353-9334Email: rmgarav...@gmail.commailto:garav...@gmail.com On May 15, 2014, at 6:50 PM, Matthew Bratkowski mab...@cornell.edumailto:mab...@cornell.edu wrote: Hello all, I am working on the structure of a small protein in space group P212121. The protein is monomeric in solution based on gel filtration analysis. The Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit results in ~50% solvent content, while 1 molecule per asymmetric unit results in ~95% solvent. I tried molecular replacement with a search model which is essentially identical in sequence to my protein, and searched for 9 or 10 molecules/asu. Using MolRep with 9 or 10 molecules/asu, I get poor contrast scores around 1-1.5. However, when using Phaser, I get a solution with one molecules/asu. Likewise, when I went back and tried MolRep with 1 molecule/asu, I got a contrast score of 3.12. This model still has some issues, but looks more correct compaired to models created with 9 or 10 molecules/asu. It seems highly unlikely that a crystal would contain 95% solvent, but is there any possiblility that this could be the case? Assuming that the Matthews coefficient is correct, does anyone have an idea why MR seems to work better for 1 molecule/asu with 95% solvent content compared to 9-10 molecules with 50% solvent content? Alternatively, is there any reason why the Matthews coefficient could be calculating incorrectly? Any
[ccp4bb] map manipulation questions
Dear All, I have a ccp4 format map file in P1 spacegroup, I would like to manipulate it in several ways: 1. enlarge the cell dimension . When I tried CELL keyword in MAPMAN, the density scaled up together with the cell dimension. Does anybody know how to do it without changing the density? 2. Change the space group to P2. 3. Move the density away from its original place, i.e. apply a translocation vector to it. Does anybody know the answers? Thanks in advance! Regards, Niu
Re: [ccp4bb] map manipulation questions
Hi Niu A long time ago I wrote a program 'maptona4' which is in CCP4. This converts a CCP4 map to and from an editable ASCII format, or at least you can edit the map header, I wouldn't advise editing the density values! So editing the cell SG is easy: just be sure to keep the numbers in the same columns. When you say apply a translocation vector do you mean (1) change the start end values but keep the density as is, or do you mean (2) keep the same start and end values and shift the density? If (1) then editing the header is the answer as before; if (2) then you'll have to find another solution. The easiest way might be to start from the MTZ file from which you obtained the map and change the phases in order to produce the shift (e.g. with sftools). Cheers -- Ian On 19 May 2014 22:25, Niu Tou niutou2...@gmail.com wrote: Dear All, I have a ccp4 format map file in P1 spacegroup, I would like to manipulate it in several ways: 1. enlarge the cell dimension . When I tried CELL keyword in MAPMAN, the density scaled up together with the cell dimension. Does anybody know how to do it without changing the density? 2. Change the space group to P2. 3. Move the density away from its original place, i.e. apply a translocation vector to it. Does anybody know the answers? Thanks in advance! Regards, Niu
Re: [ccp4bb] map manipulation questions
Niu, on 2nd thoughts, for your translocation vector, having modified the start end values in the header you could probably resample the map with extends or mapmask, provided you had a complete a.u.. Cheers -- Ian On 19 May 2014 22:25, Niu Tou niutou2...@gmail.com wrote: Dear All, I have a ccp4 format map file in P1 spacegroup, I would like to manipulate it in several ways: 1. enlarge the cell dimension . When I tried CELL keyword in MAPMAN, the density scaled up together with the cell dimension. Does anybody know how to do it without changing the density? 2. Change the space group to P2. 3. Move the density away from its original place, i.e. apply a translocation vector to it. Does anybody know the answers? Thanks in advance! Regards, Niu
Re: [ccp4bb] map manipulation questions
I found the most versatile program to manipulate maps is MAIN (http://www-bmb.ijs.si). You can copy and move maps from any cell to any other cell and get immediate visual feedback to see if things went the way you expected it. Cheers, Jens On Mon, 2014-05-19 at 17:25 -0400, Niu Tou wrote: Dear All, I have a ccp4 format map file in P1 spacegroup, I would like to manipulate it in several ways: 1. enlarge the cell dimension . When I tried CELL keyword in MAPMAN, the density scaled up together with the cell dimension. Does anybody know how to do it without changing the density? 2. Change the space group to P2. 3. Move the density away from its original place, i.e. apply a translocation vector to it. Does anybody know the answers? Thanks in advance! Regards, Niu
[ccp4bb] Reprocess data with new resolution cutoff?
Hi all, This is a basic question and I'm sure the answer is widely known, but I'm having trouble finding it. I'm working on my first structure. I have a dataset that I processed in XDS with a resolution cutoff of 2.35 A, although the data are extremely weak-to-nonexistent at that resolution limit. After successful molecular replacement and initial refinement, I then performed paired refinements against this dataset cut to various resolutions (2.95 A, 2.85 A, 2.75 A, etc). Based on the improvement in R/Rfree seen between successive pairs, it appears that the data should be cut at around 2.55 A. Here is my question: as I proceed with refinement (I'm currently using Phenix), should I now simply set 2.55 A as the resolution limit in Phenix? Or should I go back to XDS and actually reprocess the data with the new limit (2.55 A instead of 2.35 A)? Thanks, Tom
Re: [ccp4bb] Reprocess data with new resolution cutoff?
Reprocessing data to lower resolution only helps if there are ice rings or other sources of non-desired diffraction that can be eliminated as contributors to learned profiles in profile fitting. Strong ice diffraction occurs at 2.28A and 2.68A, so there is no indication that reprocessing data to lower resolution will change anything other than overall R-merge and other R-statistics. To calculate these statistics it is enough to re-merge the data with lower resolution. Zbyszek Otwinowski Hi all, This is a basic question and I'm sure the answer is widely known, but I'm having trouble finding it. I'm working on my first structure. I have a dataset that I processed in XDS with a resolution cutoff of 2.35 A, although the data are extremely weak-to-nonexistent at that resolution limit. After successful molecular replacement and initial refinement, I then performed paired refinements against this dataset cut to various resolutions (2.95 A, 2.85 A, 2.75 A, etc). Based on the improvement in R/Rfree seen between successive pairs, it appears that the data should be cut at around 2.55 A. Here is my question: as I proceed with refinement (I'm currently using Phenix), should I now simply set 2.55 A as the resolution limit in Phenix? Or should I go back to XDS and actually reprocess the data with the new limit (2.55 A instead of 2.35 A)? Thanks, Tom Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
