[ccp4bb] Crystallization problem
Hi everyone, I need an advice on some strange thing happening to one of the protein i am working on. I used to purify it and set up trays and get some needle shaped crystals and trying seeding and other methods to optimise them. But recently, it stopped giving crystals even small needles. I am still following the same protocol with same buffer stocks. And not just once but since last three times it is happening. The purified protein in gel filtration is perfectly fine eluting at same position with symmetrical distribution. However when i am setting up trays under previous conditions, i am not getting the crystals. Instead the drops are quite clear. So i increased the concentration of the protein also from 8 to 11mg/ml, but still the same. Infact i tried adding ligand also but again no crystals. So i would be really grateful if anyone can give a valuable suggestion regarding this problem !! Thanks BR Monica
Re: [ccp4bb] PHASER MR solution
Dear Jeorge, you'll find some information about this in http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Space_group_determination . A practical and easy way is described in http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Pointless HTH, Kay On Mon, 26 Jan 2015 11:24:27 +0100, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Jeorge, XDS make no claim to determine the SPACE GROUP but rather the LAUE GROUP, as only the latter is taken into account during data integration. This is definitely so during the indexing step (IDXREF.LP), but even in CORRECT, when systematic absences are sometimes indicated, XDS does not really choose the space group. Best, Tim On 01/26/2015 05:46 AM, jeorgemarley thomas wrote: Hello Dr. Randy Here is the IDXREF.LP I got in which, on the basis of quality of fit, I went for this space group well I would also try for the other screw axes. So should I Integrate the data from beginning with all possible screw axes of orthogonal space group? I am attaching the IDXREF.LP screen shot here. Jeorge On Sun, Jan 25, 2015 at 5:00 PM, Roger Rowlett rrowl...@colgate.edu wrote: Did you search all 8 possibilities of screw axes, e.g. P2221, P21212, P212121, etc? Roger Rowlett On Jan 25, 2015 4:50 AM, jeorgemarley thomas kirtswab...@gmail.com wrote: Hi, I have processed the data using XDS and space group found to be P 2 2 2 (16) and I used the phaser MR for first phase determination. The model I have used has has more than 70 % sequence identity, when I run the phaser I got the message which I have attached here. And only sum. file I got as an output. Does any one have suggestion what should I do ? I would highly appreciate your kind suggestions. Thank you in advance. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] extracting PHENIX structures (help-6435)
Thank you, Rachel. I will try this suggestion out.-Original Message-From: kra...@rcsb.rutgers.eduSent: Mon, 26 Jan 2015 09:22:00 -0700To: patrick.coss...@inbox.comSubject: Re: [ccp4bb] extracting PHENIX structures (help-6435) Dear Patrick, You can try to write a script to parse the 'software.classification' category (see http://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v40.dic/Categories/software.html) in the mmCIF file where: _software.classification = refinement and _software.name = PHENIX Please let us know if we can be of additional assistance. Sincerely, Rachel Green Rachel Kramer Green, Ph.D. RCSB PDB kra...@rcsb.rutgers.edu New! Deposit X-ray data with the wwPDB at: http://deposit.wwpdb.org/deposition (NMR and 3DEM coming soon). ___ Twitter: https://twitter.com/#!/buildmodels Facebook: http://www.facebook.com/RCSBPDB On 1/24/2015 10:41 AM, Patrick wrote: Hi, not directly related but I wanted to know how to extract from the PDB all the PHENIX/CCP4 refined structures currently deposited. Do I use the "text" search in quarry to get the result or do I use the software? I tried it for both but seems to give different results (no's). I tried for another refinement package (shelx) and same, different no's Using software, and looking for contains 320 using text search 130 Is there another way to get structure of a certain resolution ? Patrick Can't remember your password? Do you need a strong and secure password? Use Password manager! It stores your passwords protects your account. Check it out at http://mysecurelogon.com/password-manager
[ccp4bb] Reminder: Call for access to Synchrotron Beamline Facilities, 2015 - EMBL Hamburg, Germany
REMINDER: Call for access to Synchrotron Beamline Facilities, 2015 - EMBL Hamburg, Germany We would like to remind you of the approaching deadline (*Saturday 31st **January 2015*) for the call for proposals for synchrotron beamline applications at EMBL Hamburg. The call is for applications in biological small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX) for the period, April to November 2015. The following EMBL/DESY beamlines at PETRA III are available: P12 (SAXS), P13 (MX), and P14 (MX). A detailed description of the three beamlines and links to the electronic beam proposal forms can be found at: http://www.embl-hamburg.de/services/access_infrastructures/index.html The proposals will be evaluated by an external Project Evaluation Committee. Users will be informed of the results of their applications by March 16th, 2015. Access to the EMBL Hamburg facilities also includes assistance with crystallization, sample preparation and, in combination with an EMBL beamline visit, with sample characterization and optimization. Usage of the beamlines and biophysical facilities can in part be supported by the European Commission, Research Infrastructure Action under the FP7 project BioStruct-X (http://www.biostruct-x.eu/). For further general information, please contact our user office: Tel: +49 40-89902-111 Email: useroffice (at) embl-hamburg.de For specific information, please use the following email addresses: saxs (at) embl-hamburg.de (small-angle X-ray scattering) mx (at) embl-hamburg.de (macromolecular crystallography) spc (at) embl-hamburg.de (sample preparation and characterization)
[ccp4bb] Sharp: Solomon density modification step
Dear all, A quick question regarding the density modification interface via the Sharp interface. Which resolution range / radius of the solvent sphere/ ncycles should be used for optimal result? The documentation seems to suggest to restrict yourself up to the resolution where good phasing information is available (6.5A in my case) and I get excellent indicators only if I do that (they become horrible if I use the full resolution range). How about phase extension ? Which parameters would you then use? Thanks, Nicolas -- Nicolas Soler Roger Williams group MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge CB2 0QH United Kingdom phone : +44(0) 1223 26 76 20 mail : nso...@mrc-lmb.cam.ac.uk
[ccp4bb] Reminder: 3 vacancies at PDBe - closing this week
Hi all, This is just a quick reminder that the application period for our 3 vacancies is closing this Friday at midnight. We are seeking to recruit: - two curators (PDB/EMDB) - http://ig14.i-grasp.com//fe/tpl_embl01.asp?newms=jjid=53264aid=15470 - one web front-end developer - http://ig14.i-grasp.com//fe/tpl_embl01.asp?newms=jjid=53259aid=15470 Thanks, --Gerard --- Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK ger...@ebi.ac.uk . pdbe.org Secretary: Pauline Haslam pdbe_ad...@ebi.ac.uk
Re: [ccp4bb] Crystallization problem
On Mon, Jan 26, 2015 at 8:22 PM, Monica Mittal monica.mitta...@gmail.com wrote: Hi everyone, I need an advice on some strange thing happening to one of the protein i am working on. I used to purify it and set up trays and get some needle shaped crystals and trying seeding and other methods to optimise them. But recently, it stopped giving crystals even small needles. I am still following the same protocol with same buffer stocks. By 'buffer stocks', do you also mean your stock of precipitating reagent? It's happened to me that a hit I pursued and optimized vanished when I opened a new bottle of PEG. It took too long to realize that the old PEG could have been at a higher concentration than I'd assumed, given its shelf-age. I increased the PEG % and got back the crystals. Good luck, Emily. And not just once but since last three times it is happening. The purified protein in gel filtration is perfectly fine eluting at same position with symmetrical distribution. However when i am setting up trays under previous conditions, i am not getting the crystals. Instead the drops are quite clear. So i increased the concentration of the protein also from 8 to 11mg/ml, but still the same. Infact i tried adding ligand also but again no crystals. So i would be really grateful if anyone can give a valuable suggestion regarding this problem !! Thanks BR Monica
Re: [ccp4bb] Crystallization problem
Hi, It happened with me also, thought I had not the needles but the crystals. Temperature matters a lot in these situations, leave the trays unattended for long and also try with various concentration. Make it highest say 15 mg/ml and dilute serially to set with a range of concentrations. Sometime batch to batch slight change can give such situations. I would say it is a stochastic phenomenon. Best Wishes On Mon, Jan 26, 2015 at 8:22 PM, Monica Mittal monica.mitta...@gmail.com wrote: Hi everyone, I need an advice on some strange thing happening to one of the protein i am working on. I used to purify it and set up trays and get some needle shaped crystals and trying seeding and other methods to optimise them. But recently, it stopped giving crystals even small needles. I am still following the same protocol with same buffer stocks. And not just once but since last three times it is happening. The purified protein in gel filtration is perfectly fine eluting at same position with symmetrical distribution. However when i am setting up trays under previous conditions, i am not getting the crystals. Instead the drops are quite clear. So i increased the concentration of the protein also from 8 to 11mg/ml, but still the same. Infact i tried adding ligand also but again no crystals. So i would be really grateful if anyone can give a valuable suggestion regarding this problem !! Thanks BR Monica
[ccp4bb] post doc offer cryo-EM in Paris
Dear all, Please find attached a post doc offer to study cytoskeletal septins by electron microscopy. The project will be carried out at Institut curie (Paris). Someone with a former experience in electron microscopy or willing to be trained in EM is encouraged to apply. Best regards A.Bertin Aurélie Bertin Institut Curie, CNRS UMR 168 11 rue Pierre et Marie Curie, 752231 Paris cedex 05, France phone: +33 (0)1 56 24 67 82 email: aurelie.ber...@curie.fr http://umr168.curie.fr/en/Levy-group postdoc_septin.docx Description: postdoc_septin.docx
Re: [ccp4bb] PHASER MR solution
Dear Jeorge, XDS make no claim to determine the SPACE GROUP but rather the LAUE GROUP, as only the latter is taken into account during data integration. This is definitely so during the indexing step (IDXREF.LP), but even in CORRECT, when systematic absences are sometimes indicated, XDS does not really choose the space group. Best, Tim On 01/26/2015 05:46 AM, jeorgemarley thomas wrote: Hello Dr. Randy Here is the IDXREF.LP I got in which, on the basis of quality of fit, I went for this space group well I would also try for the other screw axes. So should I Integrate the data from beginning with all possible screw axes of orthogonal space group? I am attaching the IDXREF.LP screen shot here. Jeorge On Sun, Jan 25, 2015 at 5:00 PM, Roger Rowlett rrowl...@colgate.edu wrote: Did you search all 8 possibilities of screw axes, e.g. P2221, P21212, P212121, etc? Roger Rowlett On Jan 25, 2015 4:50 AM, jeorgemarley thomas kirtswab...@gmail.com wrote: Hi, I have processed the data using XDS and space group found to be P 2 2 2 (16) and I used the phaser MR for first phase determination. The model I have used has has more than 70 % sequence identity, when I run the phaser I got the message which I have attached here. And only sum. file I got as an output. Does any one have suggestion what should I do ? I would highly appreciate your kind suggestions. Thank you in advance. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] changing chain type in coot from RNA to DNA and viceversa
Hi Almudena, You can use Cootilus/Nautilus to rebuild the nucleic acid in your current density. This is not a direct conversion, but it should be quick enough. The conversion form RNA to DNA can be done (outside COOT) in a text editor by changing the residue names an deleting the O2' atoms. If you rename uridine (U) to deoxyuridine (DU), you can use 'simple mutate' in COOT to convert it into thymidine (DT). The conversion DNA to RNA is more complicated because you need to add the O2' atoms. Unfortunately, (in my hands) the fill partial residues function in COOT does not add the missing O2' atoms. You can delete and re-add the residues, but that is really slow. HTH, Robbie On 01/26/2015 10:01 AM, Almudena Ponce Salvatierra wrote: Dear all, is there a fast way of changing the chain type from RNA to DNA or from DNA to RNA within coot? I have already quite a bit built into density, but it is the wrong kind of nucleic acid. Best, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] changing chain type in coot from RNA to DNA and viceversa
Hi Robbie, thanks a lot. I was suspecting that editing the PDB was probably the easiest way to go. I will try that! Best, Almudena 2015-01-26 10:44 GMT+01:00 Robbie P. Joosten r.joos...@nki.nl: Hi Almudena, You can use Cootilus/Nautilus to rebuild the nucleic acid in your current density. This is not a direct conversion, but it should be quick enough. The conversion form RNA to DNA can be done (outside COOT) in a text editor by changing the residue names an deleting the O2' atoms. If you rename uridine (U) to deoxyuridine (DU), you can use 'simple mutate' in COOT to convert it into thymidine (DT). The conversion DNA to RNA is more complicated because you need to add the O2' atoms. Unfortunately, (in my hands) the fill partial residues function in COOT does not add the missing O2' atoms. You can delete and re-add the residues, but that is really slow. HTH, Robbie On 01/26/2015 10:01 AM, Almudena Ponce Salvatierra wrote: Dear all, is there a fast way of changing the chain type from RNA to DNA or from DNA to RNA within coot? I have already quite a bit built into density, but it is the wrong kind of nucleic acid. Best, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] (Off-topic) Analogs of CDP-choline for crystallization
Dear CCP4 members, I’m working on a phosphocholination pathway in which the enzyme utilizes the substrate Cytidine-diphosphate-choline (CDP-choline) as phosphocholione-donor. I’m enquiring about the possible analogs of CDP-choloine for cocrystallization with the enzyme as well as for the inhibition assays. Many thanks to you in advance. Have a nice day! Wenhua Zhang
[ccp4bb] changing chain type in coot from RNA to DNA and viceversa
Dear all, is there a fast way of changing the chain type from RNA to DNA or from DNA to RNA within coot? I have already quite a bit built into density, but it is the wrong kind of nucleic acid. Best, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] extracting PHENIX structures
Dear George and Patrick, It is particularly tricky to do such a search for SHELX because there are different ways of referencing it for refinement (SHELX, SHELX-76, SHELXL, SHELXL-97, SHELXL-2014 etc.) and there are other ways of using SHELX, in particular SHELXD for finding the heavy atom substructure in experimental phasing and SHELXE for improving borderline MR solutions. SHELXC/D/E are also often used in pipelines (hkl2map, ccp4i, Arcimboldo, Amber etc. and many beamline pipelines), and so may or may not be explicitely referenced in the PDB file. My private estimate of the real figure for SHELX refinement of macromolecules in 2014 would be well under 1%, but for refinement of small molecules it must be closer to 90%. Best wishes, George I did the same search as above (Patrick). I am confused how with SHELX you can get 320 (when using SOFTWARE) and then when using TEXT only 130. I get the same no's. If the word SHELX exists as follows SOFTWARE USED: SHELX the same word (SHELX) will be picked up when we do a text search as well (Its there in the remarks section under software !) But obviously NOT ! I can understand is text search gave a higher no but in this case software gives more entries. I can also understand if one PDB id is retuned for multiple searches but one keyword search (software or text) the same no must be returned. I am a bit confused in the big disparity in no's 130/320 In both cases WITHOUT software/text word for SHELX I get 552 1.0A X-ray structures of protein only. On Sun, Jan 25, 2015 at 5:57 AM, Kay Diederichs kay.diederi...@uni-konstanz.de mailto:kay.diederi...@uni-konstanz.de wrote: Two additions: a) SHELXL accounts for 16 X-Ray depositions in 2014; 2 of them also use Phenix. b) one can select Does NOT Contain: instead of Contains:. Excluding, in this manner, SHELXL, Phenix, Refmac, Buster and CNS, one gets 12 entries. These employ mostly CNX, but also coot (!), X-PLOR, PRIMEX (?), and one more REFMAC (4OM5) which for some reason does not show up in the earlier Refmac search. This shows that the results must be taken with some caution. Kay -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
Re: [ccp4bb] PHASER MR solution
Dear Jeorge, I can’t see anything in that output saying whether the systematic absences that would allow you to detect screw axes were covered in your data collection. In any case, it’s not necessary to reintegrate. All that happens when going from P222 to any one of the other 7 possible orthorhombic space groups is that some of the reflections will become systematically absent so they will be discarded, but Phaser will do that internally when testing all the space groups. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 26 Jan 2015, at 04:46, jeorgemarley thomas kirtswab...@gmail.com wrote: Hello Dr. Randy Here is the IDXREF.LP I got in which, on the basis of quality of fit, I went for this space group well I would also try for the other screw axes. So should I Integrate the data from beginning with all possible screw axes of orthogonal space group? I am attaching the IDXREF.LP screen shot here. Jeorge On Sun, Jan 25, 2015 at 5:00 PM, Roger Rowlett rrowl...@colgate.edu wrote: Did you search all 8 possibilities of screw axes, e.g. P2221, P21212, P212121, etc? Roger Rowlett On Jan 25, 2015 4:50 AM, jeorgemarley thomas kirtswab...@gmail.com wrote: Hi, I have processed the data using XDS and space group found to be P 2 2 2 (16) and I used the phaser MR for first phase determination. The model I have used has has more than 70 % sequence identity, when I run the phaser I got the message which I have attached here. And only sum. file I got as an output. Does any one have suggestion what should I do ? I would highly appreciate your kind suggestions. Thank you in advance. IDXREF.PNGSpaceG.PNG
