[ccp4bb] CCP4 BB email change
Hi, is it possible to change the email to which my ccp4i bulletin board emails get sent? The new email address is arnau.casa...@psi.ch Thank you very much, Arnau Casañas, PhD Institute of Molecular Biology and Biophysics ETH Zurich Schafmattstr. 20 HPK H10 8093 Zurich Switzerland +41.44.633845
Re: [ccp4bb] CCP4 BB email change
Hi Arnau, You can find all the information you need here: http://www.ccp4.ac.uk/ccp4bb.php Just subscribe with your new adress, and unsubscribe your old one. Best regards, Folmer ps: actually the following information is available in the header of the emails sent to the CCP4bb: List-Help: https://www.jiscmail.ac.uk/cgi-bin/webadmin?LIST=CCP4BB, mailto:lists...@jiscmail.ac.uk?body=INFO%20CCP4BB List-Unsubscribe: mailto:ccp4bb-unsubscribe-requ...@jiscmail.ac.uk List-Subscribe: mailto:ccp4bb-subscribe-requ...@jiscmail.ac.uk List-Owner: mailto:ccp4bb-requ...@jiscmail.ac.uk List-Archive: https://www.jiscmail.ac.uk/cgi-bin/webadmin?LIST=CCP4BB 2014-02-20 10:29 GMT+01:00 Casañas Arnau acasa...@mol.biol.ethz.ch: Hi, is it possible to change the email to which my ccp4i bulletin board emails get sent? The new email address is arnau.casa...@psi.ch Thank you very much, Arnau Casañas, PhD Institute of Molecular Biology and Biophysics ETH Zurich Schafmattstr. 20 HPK H10 8093 Zurich Switzerland +41.44.633845 -- Folmer Fredslund
Re: [ccp4bb] keep ligand conformation
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Koji, some ligands tend to be where the users wishes them to be. If your ligands moves away, maybe it is not really there? Maybe there is a stereochemical clash which acts more strongly than the restraints you are applying? And maybe harmonic restraints only make sense when applied to a large number of atoms, not only a few - this would depend on the implementation, though. Best, Tim On 02/20/2014 08:42 AM, Koji Yonekura wrote: Dear all, I tried to keep a conformation of a ligand during refinement with Refmac5 (Ver. 5.7.0032), and I put a harmonic restraint as, external harmonic residues from 600 A to 600 A 600 is the residue number of the ligand. I checked structures with and without the harmonic restraint, but no change was found between the two structures at all. I also tried with and without sigma weight, but nothing changed again. external harmonic restraints worked for protein parts. I would appreciate any suggestions and comments. Thank you so much in advance, Koji - -- - -- 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/ iD8DBQFTBdAOUxlJ7aRr7hoRAtNeAKDITbOxnPeTcTcoLjhOx4PLAXSmwQCfSuE1 ydSoXKpSmeFjh+WLbs32r+g= =aZu9 -END PGP SIGNATURE-
[ccp4bb] Calcium soaking
Dear all Apologies for the off-topic question: We are studying an enzyme that is activated by Ca2+. We obtained the crystals of the substrate and Ca2+-free form and solved the structure at 2.7 A resolution. However, the active site electron density map was not clear, although other regions are clear. We would like to determine the substrate-complex with Ca2+, which will elucidate the active site structure at a higher resolution. Now we have a problem that the crystals of a mutant which can bind the substrate and Ca2+ always have cracks in soaking to the crystallization solution containing CaCl2. Co-crystallization does not work at this time. We estimate the structural change in Ca2+-binding is not so big because the isozyme structure did not change very much when binding Ca2+. An isozyme structure in complex with the substrate was determined by soaking Ca2+ (and the substrate). How should we overcome this problem? Best regards ~~~ Masaki UNNO Graduate School of Science and Engineering, Ibaraki University, Japan
[ccp4bb] IYCr photo competition
The IUCr has launched a photo competition Crystallography in everyday life to celebrate the International Year of Crystallography. Photographs that capture the spirit of crystallography in the places, objects and experiences of everyday life can be submitted at http://www.iycr2014.org/participate/photo-competition. Deadline for submission is 31 May 2014. Two winners will each receive a USD 1000 bursary, sponsored by Agilent Technologies, to attend the IUCr Congress in Montreal in August 2014, where they will receive a prize certificate. Entries received by 31 March 2014 will also be considered for a Satellite Competition in Australia. The winner will receive a AUD 500 prize, sponsored by the Society of Crystallographers of Australia and New Zealand. Further information can be found at http://www.iycr2014.org/participate/photo-competition
Re: [ccp4bb] keep ligand conformation
Dear Koji, In addition to all the things Tim said, you may also have a conflict between the ligand conformation you modelled and the conformation described in your restraint file. The Refmac logfile will mark any really severe outliers, but smaller problems can still exist. Try regularising your ligand in Coot to check whether your restraints are what you want. Cheers, Robbie Sent from my Windows Phone Van: Tim Gruene Verzonden: 20-2-2014 10:51 Aan: CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] keep ligand conformation -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Koji, some ligands tend to be where the users wishes them to be. If your ligands moves away, maybe it is not really there? Maybe there is a stereochemical clash which acts more strongly than the restraints you are applying? And maybe harmonic restraints only make sense when applied to a large number of atoms, not only a few - this would depend on the implementation, though. Best, Tim On 02/20/2014 08:42 AM, Koji Yonekura wrote: Dear all, I tried to keep a conformation of a ligand during refinement with Refmac5 (Ver. 5.7.0032), and I put a harmonic restraint as, external harmonic residues from 600 A to 600 A 600 is the residue number of the ligand. I checked structures with and without the harmonic restraint, but no change was found between the two structures at all. I also tried with and without sigma weight, but nothing changed again. external harmonic restraints worked for protein parts. I would appreciate any suggestions and comments. Thank you so much in advance, Koji - -- - -- 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/ iD8DBQFTBdAOUxlJ7aRr7hoRAtNeAKDITbOxnPeTcTcoLjhOx4PLAXSmwQCfSuE1 ydSoXKpSmeFjh+WLbs32r+g= =aZu9 -END PGP SIGNATURE-
Re: [ccp4bb] Calcium soaking
Masaki, If your crystals crack when you add calcium it implies that calcium binding induces a conformational change. You should try co-crystallization with an epitaxial jump approach: Stura, E. A., Charbonnier, J.-B. Taussig, M. J.(1999) Epitaxial jumps. J. Cryst. Growth 196: 250-260. Since it is likely that calcium binding destroys only one crystal contact, you should screen for co-crystals with seeds of your calcium-free crystals. One of the planes of your calcium-free form should be able to stimulate growth of crystals with calcium but your crystallization conditions are likely to be different. Without seeding you are likely to be hindered by the nucleation barrier which seeding overcomes. Enrico. On Thu, 20 Feb 2014 11:29:47 +0100, Masaki UNNO unn...@mx.ibaraki.ac.jp wrote: Dear all Apologies for the off-topic question: We are studying an enzyme that is activated by Ca2+. We obtained the crystals of the substrate and Ca2+-free form and solved the structure at 2.7 A resolution. However, the active site electron density map was not clear, although other regions are clear. We would like to determine the substrate-complex with Ca2+, which will elucidate the active site structure at a higher resolution. Now we have a problem that the crystals of a mutant which can bind the substrate and Ca2+ always have cracks in soaking to the crystallization solution containing CaCl2. Co-crystallization does not work at this time. We estimate the structural change in Ca2+-binding is not so big because the isozyme structure did not change very much when binding Ca2+. An isozyme structure in complex with the substrate was determined by soaking Ca2+ (and the substrate). How should we overcome this problem? Best regards ~~~ Masaki UNNO Graduate School of Science and Engineering, Ibaraki University, Japan -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Symmetry problem
Dear users, thanks alot for the tips. The problem was solved! Warmest regard, Monika Coronado 2014-02-20 0:57 GMT-03:00 Jens Kaiser kai...@caltech.edu: Monika, There are several possible causes for the problem you are encountering, but your description is a little too vague to discern them. Scenario 1) You ran phaser with the option all possible spacegroups, for several different components of your crystal, setup individually, and the runs do not agree on the best spacegroup? -- In that case, phaser had problems determining the correct spacegroup, I'd suggest you search for all components, but in separate runs for each possible spacegroup. Scenario 2) You assumed your spacegroup assignment was correct, and ran MR for each of your components individually, and when you display the solutions, they overlap. In this case, you might have your solutions on different origins. The best way out is to use the first solution as a fixed solution, which is possible in most MR programs, and then search for the next component. There might be other scenarios, if you describe your situation in more detail (how many components in the crystal setup, what program you used, how you used it, and what you mean by different symmetries), we might be able to help you better, Cheers, Jens On Tue, 2014-02-18 at 14:59 -0300, Monika Coronado wrote: Dear, Does anyone know how to merge two molecules with different symmetry? I will explain: I have done the molecular replacement using the domains of the molecules separately, now I have to put all together, however they have a different symmetry. I will appreciate any kind of help. Regards, Mônika -- __o _`\,_ (*)/ (*) -+-+-+-+-+-+- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ...E tudo muda... * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * -- __o _`\,_ (*)/ (*) -+-+-+-+-+-+- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ...E tudo muda... * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
Re: [ccp4bb] Symmetry problem
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Monika, would you mind summarising how you solved your problem? It might help people with similar problems! Regards, Tim On 02/20/2014 02:45 PM, Monika Coronado wrote: Dear users, thanks alot for the tips. The problem was solved! Warmest regard, Monika Coronado 2014-02-20 0:57 GMT-03:00 Jens Kaiser kai...@caltech.edu: Monika, There are several possible causes for the problem you are encountering, but your description is a little too vague to discern them. Scenario 1) You ran phaser with the option all possible spacegroups, for several different components of your crystal, setup individually, and the runs do not agree on the best spacegroup? -- In that case, phaser had problems determining the correct spacegroup, I'd suggest you search for all components, but in separate runs for each possible spacegroup. Scenario 2) You assumed your spacegroup assignment was correct, and ran MR for each of your components individually, and when you display the solutions, they overlap. In this case, you might have your solutions on different origins. The best way out is to use the first solution as a fixed solution, which is possible in most MR programs, and then search for the next component. There might be other scenarios, if you describe your situation in more detail (how many components in the crystal setup, what program you used, how you used it, and what you mean by different symmetries), we might be able to help you better, Cheers, Jens On Tue, 2014-02-18 at 14:59 -0300, Monika Coronado wrote: Dear, Does anyone know how to merge two molecules with different symmetry? I will explain: I have done the molecular replacement using the domains of the molecules separately, now I have to put all together, however they have a different symmetry. I will appreciate any kind of help. Regards, Mnika -- __o _`\,_ (*)/ (*) -+-+-+-+-+-+- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ...E tudo muda... * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * - -- - -- 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/ iD8DBQFTBgyvUxlJ7aRr7hoRAo2BAJ0VPGwcH9FLrLI09sBwgJL9MPOVWQCgjIy1 HoLKpUi/ZtXoP5fOOMP/Kso= =44Yv -END PGP SIGNATURE-
[ccp4bb] Beamline Scientist position in XALOC beamline at the ALBA Synchrotron
Dear all, The BL13-XALOC Macromolecular Crystallography beamline at the Alba synchrotron (Barcelona) has currently an open Beamline Scientist position. We are seeking a positive, motivated scientist, with a PhD on a related topic, keen to join our beamline team in an exciting environment. Besides the continuous operation and upgrading of the beamline, the successful candidate will be encouraged to develop his/her scientific research. The beamline is equipped with state-of-the-art optics and end-station instrumentation including a high accuracy diffractometer, an automatic sample changer and a Pilatus 6M detector. XALOC started user operation in 2012, and is currently performing high-quality X-ray crystallography data collections. More info on the beamline at www.cells.es/Beamlines/XALOC. Deadline for applications is 4 March 2014. To have information on the profile and to apply to the position please refer to: http://www.cells.es/Jobs/JobOffers/ViewJob?job_id=158 Best regards, Jordi Jordi Juanhuix Gibert Experiments Division, CELLS-ALBA Synchrotron Carretera BP 1413, de Cerdanyola a Sant Cugat, km 3,3 E-08290 Cerdanyola del Vallès, Barcelona Tel: (+34) 93 592 43 22 www.cells.es http://www.cells.es/
Re: [ccp4bb] Symmetry problem
Dear Tim, I have used coot. merge molecules (calculate - merge molecules). Using coot I have moved the molecules to the same asymmetric unit. I have the same problem with two different proteins in one case it works. However, for the other I am still working :-( Greetings, Mônika 2014-02-20 11:09 GMT-03:00 Tim Gruene t...@shelx.uni-ac.gwdg.de: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Monika, would you mind summarising how you solved your problem? It might help people with similar problems! Regards, Tim On 02/20/2014 02:45 PM, Monika Coronado wrote: Dear users, thanks alot for the tips. The problem was solved! Warmest regard, Monika Coronado 2014-02-20 0:57 GMT-03:00 Jens Kaiser kai...@caltech.edu: Monika, There are several possible causes for the problem you are encountering, but your description is a little too vague to discern them. Scenario 1) You ran phaser with the option all possible spacegroups, for several different components of your crystal, setup individually, and the runs do not agree on the best spacegroup? -- In that case, phaser had problems determining the correct spacegroup, I'd suggest you search for all components, but in separate runs for each possible spacegroup. Scenario 2) You assumed your spacegroup assignment was correct, and ran MR for each of your components individually, and when you display the solutions, they overlap. In this case, you might have your solutions on different origins. The best way out is to use the first solution as a fixed solution, which is possible in most MR programs, and then search for the next component. There might be other scenarios, if you describe your situation in more detail (how many components in the crystal setup, what program you used, how you used it, and what you mean by different symmetries), we might be able to help you better, Cheers, Jens On Tue, 2014-02-18 at 14:59 -0300, Monika Coronado wrote: Dear, Does anyone know how to merge two molecules with different symmetry? I will explain: I have done the molecular replacement using the domains of the molecules separately, now I have to put all together, however they have a different symmetry. I will appreciate any kind of help. Regards, Mnika -- __o _`\,_ (*)/ (*) -+-+-+-+-+-+- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ...E tudo muda... * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * - -- - -- 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/ iD8DBQFTBgyvUxlJ7aRr7hoRAo2BAJ0VPGwcH9FLrLI09sBwgJL9MPOVWQCgjIy1 HoLKpUi/ZtXoP5fOOMP/Kso= =44Yv -END PGP SIGNATURE- -- __o _`\,_ (*)/ (*) -+-+-+-+-+-+- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ...E tudo muda... * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
Re: [ccp4bb] Symmetry problem
I thought I saw this problem before. Though I wouldn't try it if you had solutions from different space groups. http://www.phenix-online.org/documentation/find_alt_orig_sym_mate.htm F On Feb 20, 2014, at 6:09 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Monika, would you mind summarising how you solved your problem? It might help people with similar problems! Regards, Tim On 02/20/2014 02:45 PM, Monika Coronado wrote: Dear users, thanks alot for the tips. The problem was solved! Warmest regard, Monika Coronado 2014-02-20 0:57 GMT-03:00 Jens Kaiser kai...@caltech.edu: Monika, There are several possible causes for the problem you are encountering, but your description is a little too vague to discern them. Scenario 1) You ran phaser with the option all possible spacegroups, for several different components of your crystal, setup individually, and the runs do not agree on the best spacegroup? -- In that case, phaser had problems determining the correct spacegroup, I'd suggest you search for all components, but in separate runs for each possible spacegroup. Scenario 2) You assumed your spacegroup assignment was correct, and ran MR for each of your components individually, and when you display the solutions, they overlap. In this case, you might have your solutions on different origins. The best way out is to use the first solution as a fixed solution, which is possible in most MR programs, and then search for the next component. There might be other scenarios, if you describe your situation in more detail (how many components in the crystal setup, what program you used, how you used it, and what you mean by different symmetries), we might be able to help you better, Cheers, Jens On Tue, 2014-02-18 at 14:59 -0300, Monika Coronado wrote: Dear, Does anyone know how to merge two molecules with different symmetry? I will explain: I have done the molecular replacement using the domains of the molecules separately, now I have to put all together, however they have a different symmetry. I will appreciate any kind of help. Regards, Mnika -- __o _`\,_ (*)/ (*) -+-+-+-+-+-+- * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ...E tudo muda... * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * - -- - -- 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/ iD8DBQFTBgyvUxlJ7aRr7hoRAo2BAJ0VPGwcH9FLrLI09sBwgJL9MPOVWQCgjIy1 HoLKpUi/ZtXoP5fOOMP/Kso= =44Yv -END PGP SIGNATURE-
[ccp4bb] Recovering crystals from dry drops
Would you please share your experience and comments on recovering protein crystals from dry (or almost dry hanging drops) for data collection. I found some beautiful crystals in hanging drops that were set up three years ago; from the color of the crystals ( the protein binds a colored substrate) and the their shape I know these are crystals of my protein. I had collected data using some of these crystals when they were fresh; resolution was poor and the overall I/sigma was low. I am curious if the dehydration would improve the diffraction now. Thanks Debasish Ph: (205)934-0124; Fax: (205)934-0480
Re: [ccp4bb] Recovering crystals from dry drops
Hi Debasish, I would first use some of those crystals to make seeds and grow some new crystals so that I would not lose the crystal. Dehydration, even done systematically (eg, http://www.mitegen.com/mic_catalog.php?c=jenCrystaloptdehydrate), may or may not improve the diffraction. Like most other things in biological crystallography, it varies from case to case. I do not think other people’s experience really means anything for your crystals. Zhijie From: Debasish Chattopadhyay Sent: Thursday, February 20, 2014 10:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Recovering crystals from dry drops Would you please share your experience and comments on recovering protein crystals from dry (or almost dry hanging drops) for data collection. I found some beautiful crystals in hanging drops that were set up three years ago; from the color of the crystals ( the protein binds a colored substrate) and the their shape I know these are crystals of my protein. I had collected data using some of these crystals when they were fresh; resolution was poor and the overall I/sigma was low. I am curious if the dehydration would improve the diffraction now. Thanks Debasish Ph: (205)934-0124; Fax: (205)934-0480
Re: [ccp4bb] Recovering crystals from dry drops
Debasish Assuming you do not need seeds, just lift the coverslip, add a small amount of water to the reservoir, close and allow the drop to equilibrate for 20min, or untill you are sure that you have enough liquid to avoid that the drops becomes solid while you pick up the crystals with a loop and transfer to a suitable cryosolution very rich in cryocomponents with very little water. Improved diffraction? 10% chance! Enrico. On Thu, 20 Feb 2014 17:21:53 +0100, Zhijie Li zhijie...@utoronto.ca wrote: Hi Debasish, I would first use some of those crystals to make seeds and grow some new crystals so that I would not lose the crystal. Dehydration, even done systematically (eg, http://www.mitegen.com/mic_catalog.php?c=jenCrystaloptdehydrate), may or may not improve the diffraction. Like most other things in biological crystallography, it varies from case to case. I do not think other people’s experience really means anything for your crystals. Zhijie From: Debasish Chattopadhyay Sent: Thursday, February 20, 2014 10:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Recovering crystals from dry drops Would you please share your experience and comments on recovering protein crystals from dry (or almost dry hanging drops) for data collection. I found some beautiful crystals in hanging drops that were set up three years ago; from the color of the crystals ( the protein binds a colored substrate) and the their shape I know these are crystals of my protein. I had collected data using some of these crystals when they were fresh; resolution was poor and the overall I/sigma was low. I am curious if the dehydration would improve the diffraction now. Thanks Debasish Ph: (205)934-0124; Fax: (205)934-0480 -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] identifying protein crystals via visible light only?
Hi Richard and All, At Formulatrix we haven't yet seen an algorithm that can come even close to what the human brain can do for drop scoring. Crystals come in too many shapes, sizes, plates, and confusing backgrounds making automated detection extraordinarily difficult. Because of this, we have found users will not trust or use an automated scoring algorithm for visible light. Plus, these algorithms usually only reliably pick out the easy crystals which a human can usually do in a flash by just looking at 96 thumbnails. If there is a an algorithm out there that is successful with visible light crystal detection, we would love to hear about it. Indeed, as Jose and Zhijie points out, when you factor in additional detection technologies like UV and SHG (Second Harmonic Generation a.k.a. SONICC) then image processing can start to be helpful. In this case, however, Formulatrix only uses image processing to resort the images to put drops most likely to contain crystals at the top of the list. We call this auto scoring but it's really auto sorting. We never pitch this as infallibly able to score drops, because it certainly isn’t. Interestingly, the additional information from UV or SHG combined with auto scoring doesn't necessarily reduce the time looking at drops. Yes, you can find the easy crystals much faster with these techniques. But, what we see instead is we are giving users even more information to look through and the advantage isn’t a time savings for looking at drops. Instead, you find smaller crystals and find crystals you otherwise would have missed. This gives you more hits that you can optimize sooner. We would love to hear further opinions, especially contrarian, from the community on this topic.
[ccp4bb] identifying protein crystals via visible light only?
Well, Ellen, the opinion of us in Agilent is that if you're wanting to make a judgement of the diffraction qualities (resolution limit, mosaicity and unit cell, etc.) of (putative) protein crystals, in situ, then don't bother with visible light at all: instead use X-rays !! For this reason, we've developed the PX Scanner system. With the development of ever brighter microfocus sources, increasingly sensitive / extremely low noise CCD area detectors and the availability of 'X-ray friendly' crystallisation plates, the capability of the PX Scanner for high throughput crystal screening work has never been higher. All are Welcome. Best Regards, Marcus Winter (Agilent Technologies) http://www.chem.agilent.com/en-US/products-services/Instruments-Systems/X-Ray-Crystallography/PX-Scanner/Pages/default.aspx -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ellen Gualtieri Sent: Thursday, February 20, 2014 5:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] identifying protein crystals via visible light only? Hi Richard and All, At Formulatrix we haven't yet seen an algorithm that can come even close to what the human brain can do for drop scoring. Crystals come in too many shapes, sizes, plates, and confusing backgrounds making automated detection extraordinarily difficult. Because of this, we have found users will not trust or use an automated scoring algorithm for visible light. Plus, these algorithms usually only reliably pick out the easy crystals which a human can usually do in a flash by just looking at 96 thumbnails. If there is a an algorithm out there that is successful with visible light crystal detection, we would love to hear about it. Indeed, as Jose and Zhijie points out, when you factor in additional detection technologies like UV and SHG (Second Harmonic Generation a.k.a. SONICC) then image processing can start to be helpful. In this case, however, Formulatrix only uses image processing to resort the images to put drops most likely to contain crystals at the top of the list. We call this auto scoring but it's really auto sorting. We never pitch this as infallibly able to score drops, because it certainly isn’t. Interestingly, the additional information from UV or SHG combined with auto scoring doesn't necessarily reduce the time looking at drops. Yes, you can find the easy crystals much faster with these techniques. But, what we see instead is we are giving users even more information to look through and the advantage isn’t a time savings for looking at drops. Instead, you find smaller crystals and find crystals you otherwise would have missed. This gives you more hits that you can optimize sooner. We would love to hear further opinions, especially contrarian, from the community on this topic.
Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein
Thanks to everyone who responded to my post (especially, John Lee and Brad Bennett) with helpful comments and suggestions. The good news is that I was able to get the Ulp1 protease cleavage step to 100% completion at 4C by using between 1:3-to-1:2 enzyme:substrate ratios and incubating the reactions overnight on an orbital shaker to ensure constant mixing (instead of dialysis). It turns out that I didn't have to switch from beta-mercaptoethanol to TCEP. I also kept NaCl concentration at 150mM Nacl and that was okay as well. Many thanks that I can now be off to the next hurdle! Raji On Sun, Feb 16, 2014 at 10:58 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] SBGrid/NE-CAT Computing School - Registration Open
Please join us for the SBGrid/NE-CAT Computing School: Quo Vadis Structural Biology 2014? Data Processing in Crystallography SBGrid/NE-CAT Computing School June 5-7th, 2014 Harvard Medical School, Boston, MA Registration Fee: $200 Register herehttp://www.events.harvard.edu/profile/web/index.cfm?PKWebId=0x129021057 . Scientific Advisory Board: Axel Brunger, Lynne Howell, Yuh Min Chook Organizing Committee: David Neau, Jon Schuerman, Piotr Sliz Sponsors: DECTRIS, Bruker AXS, Inc. Two nanocourse credits will be issued to eligible Harvard graduate students. SBGrid Certificates will be issued to all participants who attend both days of the conference and present a poster. Bruker Poster Awards will be given to attendees for the top three poster presentations. Thursday, June 5 - Day 1: Nanocourse I - Data Processing 9-12:20: Introduction to data processing - Jim Pflugrath: Indexing and Integration (50 mins) - Zbyszek Otwinowski: Scaling and merging (50 mins) - coffee break (15 mins) - Kay Diederichs: CC* - Linking crystallographic model and data quality (50 mins) - Panel Discussion: How to present your data statistics in Table I (20 mins) - Piotr Sliz: Data processing and archiving tools in the SBGrid Collection (15 mins) 2-5 PM Practical Session (3h): Analyzing application outputs for conventional and onerous data sets - Phil Evans: iMosflm/pointless and aimless (45 mins) - Zbyszek Otwinowski: HKL3000 (45 mins) - coffee break (15 mins) - Kay Diederichs: XDS (45 mins) - Matt Benning: Processing Twinned and split crystals with Rlatt - (30 mins) 5-7PM - Poster Session/Reception Friday, June 6 - Day 2: Nanocourse II - Data Collection and Processing Fast Lane 9-12 Collecting data at third generation beamlines - Jon Schuermann: Optimizing Data Collection with micro-diffraction tools (40 mins) - Raj Rajashankar: Dose-sliced data collection (40 mins) - coffee break (20 mins) - Clemens Schulze-Briese: Data collection with PAD detectors (40 mins) - David Neau: Getting the most from remote data collection (40 mins) 1:30-2:30PM: Recent advances in data processing pipelines - Nick Sauter: DIALS: Advanced data reduction (30 mins) - Jon Schuermann: RAPD (30 mins) 2:45 - 4:15PM: Special Topics - Dominika Borek: Last chance strategies in data processing (45 Min) - Nick Sauter: cctbx.xfel: New software for serial crystallography (45 mins) 4:15-10PM Meet with Experts/Reception (diffraction images required!) Saturday, June 7 - Day 3: Structural Biology Symposium *SESSION DETAILS COMING SOON* - Morning: Structural biology talks by invited speakers and trainees - Afternoon: Phenix presentations by Paul Adams, Tom Terwilliger, and Nat Echols
Re: [ccp4bb] Recovering crystals from dry drops
Hi Debasish, I had a go at fishing crystals out of dried-out sitting drops some time ago: The crystals were retrieved from the surrounding viscous liquid by dropping 1 µl cryoprotectant over the drop. This immediately started to thin the liquid, freeing the crystals within, so that one could be scooped out using a nylon loop mounted on a metal pin. The crystal was then immediately frozen in liquid nitrogen. The crystals started to dissolve in the cryoprotectant if left longer than ~1 min, so there was generally only time to retrieve one crystal from any one drop. The crystals diffracted to 2.1 A. Sharpe, Baker and Lott. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 June 1; 61(Pt 6): 565--568. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1952335/ Good luck, Miriam On 21/02/14 4:59 AM, Debasish Chattopadhyay wrote: Would you please share your experience and comments on recovering protein crystals from dry (or almost dry hanging drops) for data collection. I found some beautiful crystals in hanging drops that were set up three years ago; from the color of the crystals ( the protein binds a colored substrate) and the their shape I know these are crystals of my protein. I had collected data using some of these crystals when they were fresh; resolution was poor and the overall I/sigma was low. I am curious if the dehydration would improve the diffraction now. Thanks Debasish Ph: (205)934-0124; Fax: (205)934-0480
Re: [ccp4bb] Recovering crystals from dry drops
I recently had a similar situation for a protein crystal that was sitting in very thick, sticky goop. I put three drops of Paratone N on a coverslip and harvested the crystal by using a Mitegen yoke tool to dig around the crystal, then a regular nylon loop to move the crystal into a drop of Paratone N. A bit of the semi solid goop came over with the crystal, but I was able to swish it away in the Paratone N. I repeated this in a total of three drops and then flash froze it in liquid nitrogen. The crystal diffracted to better than 2 Å at the synchrotron. Good luck on recovering your crystals! Regards, Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish Chattopadhyay Sent: Thursday, February 20, 2014 11:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Recovering crystals from dry drops Would you please share your experience and comments on recovering protein crystals from dry (or almost dry hanging drops) for data collection. I found some beautiful crystals in hanging drops that were set up three years ago; from the color of the crystals ( the protein binds a colored substrate) and the their shape I know these are crystals of my protein. I had collected data using some of these crystals when they were fresh; resolution was poor and the overall I/sigma was low. I am curious if the dehydration would improve the diffraction now. Thanks Debasish Ph: (205)934-0124; Fax: (205)934-0480 -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
[ccp4bb] XDS vs SADABS absorption correction factors
All, Sorry, this is a little bit off topic. I could not find a thorough definition of the Correction Factors for absorption correction in XDS nor of the Transmittance factors in SADABS. We collected small molecule samples on a synchrotron beamline, processed the data with XDS and the user now needs to know the Minimum and Maximum T for their cif. From what I could gather, the XDS correction factors in the ABSORP.CBF are multiplied by 1000; the values are around 1 (i.e a little smaller /and/ a little larger). The cif dictionary for _exptl_absorpt_correction_T_ states The permitted range is 0.0 - 1.0, which clearly has to be a different definition. Any pointers are welcome, Cheers, Jens
