[ccp4bb] error when running pandda.inspect
Dear community, I am trying to run panDDA. As first, I am simply trying to follow the tutorial. I have an issue with pandda.inspect: "INFO:: There are 1 command line scripts to run /storage/software/ccp4/ccp4-8.0/bin/../lib/python3.7/site-packages/pandda/inspect/__init__.py calling run_script() for file /storage/software/ccp4/ccp4-8.0/bin/../lib/python3.7/site-packages/pandda/inspect/__init__.py debug:: run_script() on /storage/software/ccp4/ccp4-8.0/bin/../lib/python3.7/site-packages/pandda/inspect/__init__.py Running python script /storage/software/ccp4/ccp4-8.0/bin/../lib/python3.7/site-packages/pandda/inspect/__init__.py Traceback (most recent call last): File "", line 1, in File "/storage/software/ccp4/ccp4-8.0/bin/../lib/python3.7/site-packages/pandda/inspect/__init__.py", line 1, in import giant.logs as lg ImportError: No module named giant.logs" So I tried to fixed it myself, without success up to now. Maybe I am missing something obvious. I did search on the ccp4bb archive. Someone had exactly the same issue. But I couldn't find any answer. If someone could help me. Have a nice day. Nicolas -- Nicolas Foos PhD - ARISE fellow https://orcid.org/-0003-2331-8399 EMBL Grenoble, McCarthy Team 71 av. des Martyrs, 38000 Grenoble FRANCE +33 4 57 42 84 67 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Crystals with DNA
Hello, If you want to promote "infinite" helix, you should go for overhangs (sticky end) with compatible sequence. I try to explain better what I have in mind (and I actually did). example : 5'ATCCCTAAATCGGCGTGTGCT---3' 3'---GGATTTAGCCGCACACGATAG5' Hoping that this results in something like : 5' ATCCCTAAATCGGCGTGTGCTATCCCTAAATCGGCGTGTGCTATCCCTAAATCGGCGTGTGCT---3' 3' ---GGATTTAGCCGCACACGATAGGGATTTAGCCGCACACGATAGGGATTTAGCCGCACACGATAG5' With the blunt end, you may have the helix, or not, in my opinion your are not really promoting the "infinite helix". Nicolas On 09/02/2024 10:59, careinaedgo...@yahoo.com wrote: Thank you for this insight, Nicolas. It is very helpful. Yes I have also had a soccer ball shaped crystal that does not diffract as well as, and more recently, many plate like crystals but they do not diffract either. I do know I have both protein and DNA in my crystals but I do not know, as you say, exactly what is forming the crystal contacts. Just to be clear, do you say overhangs are helpful? Surely overhangs won't promote an infinite helix? If one wants an infinite helix, would the DNA not have to be blunt ended? Sent from Yahoo Mail on Android <https://mail.onelink.me/107872968?pid=nativeplacement=Global_Acquisition_YMktg_315_Internal_EmailSignature_sub1=Acquisition_sub2=Global_YMktg_sub3=_sub4=10604_sub5=EmailSignature__Static_> On Thu, Feb 8, 2024 at 4:59 PM, Nicolas Foos wrote: Hello Careina, In my hands, DNA protein complex crystals may be frustrating, because often we get good looking crystals which don't diffract at all and are actually not easy to improve. I remember obtaining a lot of crystal looking a bit like "STOP" road sign (octogonal shape for one axis) which never diffracts. (Often containing only DNA not well organized) So long story short, In my hand (transciption factor bound with homeodomain for example). I had good results with DNA sequence which results in hoverhangs. The idea was to bet on a "infinit" DNA helix which should help the packing. I strongly encouraged you to rely on any other information you can have to be sure of what is the best minimal sequence (like band shift assay). Also if you can purify the entire complex before crystallization assay (I don't know your protocol, but ideally, I would prepare the complex prot-DNA and put it on size exclusion). The point is, you don't know /a priori /what kind of crystal packing you will have. It may be only due to protein protein contact and not related to the DNA directly. Also, I often get good results with crystal growing condition containing MPD or PEG (makes me using PEG screen Familly as first approach). I invite you to read the Timothy Richmond teams Papers on nucleosome they spend some times improving the resolution on very large complex. (Luger etal 1997). There is many parameters, DNA sequence also change a bit the DNA geometry (look for A-tract), You may want to introduce such sequence to maybe improve the "rigidity". Also if your DNA fragment are small, be careful with the temperature. The annealing and the DNA duplex formation is critical and you should be careful on your procedure. I remember that small cation like Li, may help too. HTH Nicolas On 08/02/2024 12:25, careinaedgo...@yahoo.com <mailto:careinaedgo...@yahoo.com> wrote: Hello all. I am struggling to get defracting crystals with a protein DNA complex. The crystals are plentiful but they do not diffract. I am going back to the grind stone and relookong at my DNA sequence. Is there any wisdom you could give me with regards to what works best with DNA in crystals? From my reading it seems if the length is a multiple of 7 (for B DNA) and blunt ended, it will stretch over the length of the crystal and improve crystalisability. But if you want crystals that diffract better, you will need to play with length and even making it only one base longer or shorter can make a difference, even changing the morphology of the crystal? Longer is better than shorter, and overhangs are good for improving diffraction? Presumably because they stabilize contacts? It is expensive to synthesize a while bunch of sequences so I need to be strategic in my choice. Would appreciate any advice. Thank you Careina. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1> -- Nicolas Foos PhD - ARISE fellow https://orcid.org/-0003-2331-8399 <https://orcid.org/-0003-2331-8399>
Re: [ccp4bb] Crystals with DNA
Hello Careina, In my hands, DNA protein complex crystals may be frustrating, because often we get good looking crystals which don't diffract at all and are actually not easy to improve. I remember obtaining a lot of crystal looking a bit like "STOP" road sign (octogonal shape for one axis) which never diffracts. (Often containing only DNA not well organized) So long story short, In my hand (transciption factor bound with homeodomain for example). I had good results with DNA sequence which results in hoverhangs. The idea was to bet on a "infinit" DNA helix which should help the packing. I strongly encouraged you to rely on any other information you can have to be sure of what is the best minimal sequence (like band shift assay). Also if you can purify the entire complex before crystallization assay (I don't know your protocol, but ideally, I would prepare the complex prot-DNA and put it on size exclusion). The point is, you don't know /a priori /what kind of crystal packing you will have. It may be only due to protein protein contact and not related to the DNA directly. Also, I often get good results with crystal growing condition containing MPD or PEG (makes me using PEG screen Familly as first approach). I invite you to read the Timothy Richmond teams Papers on nucleosome they spend some times improving the resolution on very large complex. (Luger etal 1997). There is many parameters, DNA sequence also change a bit the DNA geometry (look for A-tract), You may want to introduce such sequence to maybe improve the "rigidity". Also if your DNA fragment are small, be careful with the temperature. The annealing and the DNA duplex formation is critical and you should be careful on your procedure. I remember that small cation like Li, may help too. HTH Nicolas On 08/02/2024 12:25, careinaedgo...@yahoo.com wrote: Hello all. I am struggling to get defracting crystals with a protein DNA complex. The crystals are plentiful but they do not diffract. I am going back to the grind stone and relookong at my DNA sequence. Is there any wisdom you could give me with regards to what works best with DNA in crystals? From my reading it seems if the length is a multiple of 7 (for B DNA) and blunt ended, it will stretch over the length of the crystal and improve crystalisability. But if you want crystals that diffract better, you will need to play with length and even making it only one base longer or shorter can make a difference, even changing the morphology of the crystal? Longer is better than shorter, and overhangs are good for improving diffraction? Presumably because they stabilize contacts? It is expensive to synthesize a while bunch of sequences so I need to be strategic in my choice. Would appreciate any advice. Thank you Careina. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1> -- Nicolas Foos PhD - ARISE fellow https://orcid.org/-0003-2331-8399 EMBL Grenoble, McCarthy Team 71 av. des Martyrs, 38000 Grenoble FRANCE +33 4 57 42 84 67 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Fragile Crystals
Dear Morgan Elizabeth, To complete the answers and suggestion made by the ccp4 community, I would add, that you can actually combine the service offered by HTX facility : You send your protein, and they are preparing the crystallization plate for you in CD-plate which may be use in both Harvester and for Direct in-situ data collection (no harvesting, nothing except the X-ray will touch the crystals). The great advantage is that like this, you don't have to be worried by the plate transportation. They are prepared on-site and then if you go for an in-situ experiment, it can be measured on three of the ESRF beamlines. Notably, we can on MASSIF-1 combine in-situ screening then if required automated harvesting directly at the beamline. Feel free to contact us. mbow...@embl.fr didier.nuri...@esrf.fr marq...@embl.fr All the best, Nicolas On 23/11/2023 10:31, Jose A. MARQUEZ wrote: Dear Elizabeth, /In situ/ data collection is a good approach to try in your case. You could use the CrystalDirect technology for automated crystal harvesting that is more gentle to crystals than manual harvesting. This is available both at EMBL Grenoble and EMBL Hamburg facilities, which offer integrated crystallography services in collaboration with the ESRF and Petra III synchrotrons. doi:10.3791/62491. doi:10.1016/j.crmeth.2021.100102. doi:10.1107/s0907444912031459. Best wishes Josan _ Jose A. Marquez, Senior Scientist Head of the Crystallization Facility European Molecular Biology Laboratory, Grenoble. Delivery address: EMBL, 71, Avenue des Martyrs 38000 Grenoble, France Postal address: EMBL, 71, Avenue des Martyrs CS 90181 38042 Grenoble Cedex 9, France Phone +33 (0)476 20 74 25 Fax. +33 (0)476 20 71 99 https://www.embl.org/groups/marquez/ https://www.embl.org/services-facilities/grenoble/high-throughput-crystallisation/ https://htxlab.embl.org/ _ On 11/22/2023 5:44 PM, Blake, Morgan Elizabeth wrote: Hello! I am a PhD student working on a crystallography project to wrap up my dissertation research. I have purified a complex of two proteins, and I can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 7.5. These crystals have sharp edges and can grow to a large size (greater than 0.5 mm), but the crystals seem to be very fragile. When we open the drops to harvest the crystals, we have little time to harvest the crystals before they crack. When we move the crystals to a cryoprotectant, over time they start fracturing. We've tried using different percentages of glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, the crystals do not diffract well, with spot patterns that look very streaky/mosaic, which I presume is due to the defects that we see in harvesting/handling. We have screened for alternate crystallization conditions, but we seem to get the same morphology in other conditions. Does anyone have suggestions for additives we could use post-crystallization to help stabilize our crystals? Thanks for your advice! To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1> -- Nicolas Foos PhD - ARISE fellow https://orcid.org/-0003-2331-8399 EMBL Grenoble, McCarthy Team 71 av. des Martyrs, 38000 Grenoble FRANCE +33 4 57 42 84 67 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Comparing two datasets
Hi Mirek, I am pretty sure XSCALE will do that for you : https://xds.mr.mpg.de/html_doc/xscale_program.html If not, maybe have a look on SHELXC in SIR mode. Hope this help. Nicolas On 25/07/2022 21:52, Cygler, Miroslaw wrote: Hi, I would like to calculate the R-merge for Fs from two datasets processed from two different crystals. Tried to use Blend but got the message that Blend requires R. Downloaded R but do not know how to tell CCP4 where it is located on my Mac. Is there another program that would take two mtg files and merge the Fs? Any help would be greatly appreciated. Mirek To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1> -- Nicolas Foos PhD - ARISE fellow https://orcid.org/-0003-2331-8399 EMBL Grenoble, McCarthy Team 71 av. des Martyrs, 38000 Grenoble FRANCE +33 4 57 42 84 67 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] MR solution not working
Hello, I have a maybe naive question, did you check the results for the Matthew Probabilities calculation ? Are you 100% certain that's your protein of interest in the crystal? Sometimes we can have surprise. Nicolas On 03/03/2022 05:43, Shubhashish Chakraborty wrote: Hello, I am trying to solve a dataset using molecular replacement. However, neither Phaser MR nor Molrep can give any solution. In Phaser, I have received an advisory that Top FTF has not packed. I have tried molecular replacement using the wild-type protein at different resolutions (I am working on a mutant). Also, I have truncated the loops from the input structure. However, none have worked. So, what can be the possible way to solve this data set? Thank you Shubhashish Chakraborty PhD JRF 2018 Structural and Molecular Biology Lab (Varma Lab) Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) Khargar, Navi Mumbai E-mail: schakrabo...@actrec.gov.in <mailto:schakrabo...@actrec.gov.in> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1> -- Nicolas Foos PhD - ARISE fellow https://orcid.org/-0003-2331-8399 EMBL Grenoble, McCarthy Team 71 av. des Martyrs, 38000 Grenoble FRANCE +33 4 57 42 84 67 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] scale and merge suggestions
Hi Almudena, did you cut the res. after the merge of your "individual" datasets ? or you cut for each and then you merged? Because I will probably go for the first, I merge and then I decide where to cut. If you cut for each, maybe you "miss" the gain provide by the redundancy you have cumulated using all the data. If I mis understood what you did, and return a bad answer, sorry. And to really answer your question, I will say : I stick to a combination of the "standard" stats, CC 1/2 mainly, I/sig and if you expect anomalous, CC Ano and SigAno. My choice would be conditioned also by the strategy for the phasing. Hope to help. De: "Almudena Ponce Salvatierra" À: "CCP4BB" Envoyé: Mercredi 4 Novembre 2020 17:30:29 Objet: [ccp4bb] scale and merge suggestions Hello everyone, I have collected 6 small datasets from a crystal, each from a different point on the crystal to avoid radiation damage, and there is some overlap among them, as follows: - dts1: 0-90 degrees - dts2: 60-150 degrees - dts3: 120-210 degrees - dts4: 180-270 degrees - dts5: 240-330 degrees - dts6: 300-390 degrees Diffraction is very poor and, after processing, I've cut resolution at around 5 Angstroms for all of them. The space group is C2. Feeding the 6 of them to scale_and_merge in Phenix gives the attached output. Does anybody who has merged such low-resolution anomalous data before know whether this is a "good" indicator (meaning there's a tiny light at the end of the tunnel) or rather not? Any suggestions? (besides the obvious "go back to the lab and grow new crystals") My aim is to combine these phases with a partial molecular replacement solution and a native dataset at 3.5 Angstroms. Hopefully, then I would be able to finish building the model. Thanks a tone in advance! Best, Almudena To unsubscribe from the CCP4BB list, click the following link: [ https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 | https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 ] To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] best program for merging the two datasets
Hi Kahkashan, did you consider to use ccCluster. This program is very helpful. It's designed exactly for what you want. It allow an easy and quick analysis of which data sets you should merge. You can find it here : [ https://github.com/gsantoni/ccCluster | https://github.com/gsantoni/ccCluster ] And the paper : [ https://journals.iucr.org/j/issues/2017/06/00/ap5019/ | https://journals.iucr.org/j/issues/2017/06/00/ap5019/ ] in open access. Hope this help. Nicolas De: "Firdous Tarique" À: CCP4BB@JISCMAIL.AC.UK Envoyé: Mercredi 15 Janvier 2020 15:08:29 Objet: [ccp4bb] best program for merging the two datasets Hi. I have collected multiple datasets for my crystals and now want to merge them. Iwanted to know that between Blend and pointless, which programme is better to merge two or more data sets? Thanks Kahkashan To unsubscribe from the CCP4BB list, click the following link: [ https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 | https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 ] To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] unmounted cryoloops supplier
Dear Simone, I think here is what you are looking for : https://www.mitegen.com/product/dual-thickness-micromounts/ Best, Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 14/05/2019 14:20, De Rose, Simone wrote: Dear All, I’m looking to buy some loose/unmounted cryoloops in strip (see link below) but it seem that all the well know suppliers doesn’t sell those anymore. I could only find mounted ones. Does anyone know a supplier for a similar product? https://www.moleculardimensions.com/applications/upload/Litholoops_2011.pdf Best regards Simone *Simone Antonio De Rose* Postdoctoral Research Associate University of Exeter www.exeter.ac.uk Henry Wellcome Building for Biocatalysis, Stocker rd, EX44QD, Exeter, UK http://www.exeter.ac.uk/codebox/email-sig/staff-sig.gif This email and any attachment may contain information that is confidential, privileged, or subject to copyright, and which may be exempt from disclosure under applicable legislation. It is intended for the addressee only. If you received this message in error, please let me know and delete the email and any attachments immediately. The University will not accept responsibility for the accuracy/completeness of this email and its attachments. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Ramachandran outliers
Dear Tereza, In certain cases it could be better to do a step back to be able to rebuild properly. Did you look carefully in the real-space the agreement between your model and the visible density. If you "over"refined your model in reciprocal space only you can loose some information. I comment you last comments : 1) Density too weak. Was it like that from the beginning, what happen if you remove the "invisible" parts, is any differences green or red density visible in the vicinity? Is that a flexible loop ? The "systematic" coincidence between weak density and ramachandran outlier is suspicious. 2) Manual NCS definition could help in a first approach but once your model is complet enough it could be good to relax this constrain, because you are actually fitting the model with what you think it should be and what it actually is. In my opinion it's interesting to at least try to use automatic which maybe will keep as NCS only the parts which are really NCS. Sometimes very similar conformation of domain or sub-units are close to be an NCS but not anymore NCS because already too different (I am not sure to be clear ;-) ). 4) pdb redo even if really powerful and efficient can't be magic, if your model suffer from mistake such as really bad rotamer or ramachandran outlier, it could be impossible to revert that. Only a manual intervention could fix it. If the ramachandran outlier are in a weak density area, I would probably try to fix that localy based on geometrical constraint (included ramachandran) and then redo a round of refinement in reciprocal space to see what happen. Sometimes the distortion is that important that you need to re-build more than the residues directly involved especially at relatively "low" resolution. Hope this help. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 08/03/2019 10:36, Tereza Skalova wrote: Thank you for your comments. 1) manual correction in Coot does not work - the density is too weak 2) manual NCS is substantially better than automatic local NCS in this case 3) CPP4i2 might be good idea 4) PDB REDO is great, however no more help in this case 5) Prosmart - I use "prosmart -id -p1 target.pdb -p2 external.pdb" , I will study other possibilities Tereza pá 8. 3. 2019 v 10:25 odesílatel Robert Nicholls mailto:nicho...@mrc-lmb.cam.ac.uk>> napsal: Dear Tereza, It is highly recommended that you do not attempt to directly optimise the Ramachandran plot during refinement. Doing so would not guarantee you a better model, and would mean that you could no longer use the Ramachandran plot for validation purposes. I suggest that you inspect each of the residues corresponding to the outliers, and assess the conformation, geometry and density fit of that residue and residues in the surrounding region. There are various tools in Coot to help you with this. It should be clear which regions are in need of attention (outliers that should be fixed) and which "outliers" should be considered acceptable. Indeed, ensuring that there are no Ramachandran outliers is not an objective/requirement for a good model. I refine in Refmac, using h-bond based Prosmart restraints based on PDB structures (identical molecules with high resolution) Prosmart h-bond based restraints, and Prosmart restraints based on PDB structures are two different things. Are you using Prosmart h-bond restraints, or Prosmart restraints to a high-resolution homologous model? If you're using restraints to a high-resolution homologue, are you generating restraints for all of your chains, or just some of them? If you're just generating restraints for some of them, then you should ensure that the others are appropriately restrained also. I use NCS, medium between AB (protein 1) and loose between CDE (protein 2). From your mention of "medium" and "loose" NCS restraints, I'm guessing you're using CCP4i. Why not try using CCP4i2? This is the currently recommended and supported interface for CCP4 software. Don't use manual NCS restraints (medium, loose, etc.). Try using automatically generated local NCS restraints - we find they work better. How are your R/Rfree behaving in refinement? If you're using Prosmart restraints to homologous models then do you need/benefit from the use of NCS restraints too, or are they working against each other? Best regards, Rob On 8 Mar 2019, at 09:08, Tereza Skalova mailto:t.skalova.c...@gmail.com>> wrote: Dear all, I have structure at 3.3A resolution and I have ca. 35 Ramachandran outliers. Do you have any idea how to reduce
Re: [ccp4bb] Python3 and MTZ
Dear Kay, depending of the motivation to develop in python3 (could be due to an OS using python3 by default or you really prefer to work with python3). If it's due to the OS, a possible strategy is to use virtualenv (https://virtualenv.pypa.io/en/stable/) which let you use python2 even if python3 is the default version for the OS. It exist probably other method to have a contain installation of python2 with all the library needs. I used this strategy (virtualenv) to install ccp4 (with the installer which needed python2) on a manjaro linux (Arch based) running python3 and that works very well. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 06/06/2018 14:25, Kay Diederichs wrote: Dear all, I haven't tried to read MTZ files from Python until now, but for a new project in my lab I'd like to do that - and with Python3. Googling around, it seems that iotbx from cctbx is not (yet) Python3-compatible. So, what are my options? thanks, Kay To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] merging an hkl file
I lkije the riddle, so: I guess it's something for Signal Noise Ratio (?) (help by the context) ;-) Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 18/04/2018 11:29, Eleanor Dodson wrote: What does snr mean? Eleanor On 18 April 2018 at 00:27, Gihan Ketawala <gketa...@asu.edu <mailto:gketa...@asu.edu>> wrote: can someone tell me what are the posible consiquences of merging only the reflection are snr >= 1 ?
Re: [ccp4bb] How to fit DNA in density map of protein-DNA complex
Hi Madhusudhan, As already mentioned, coot is perfectly adapted. But if you have a pretty long DNA strand, maybe you can design an "artificial" DNA strand with http://structure.usc.edu/make-na/server.html It generated different form of DNA (B, Z and A). You can use your known sequence and create a pdb model. Once you have this, you can fit it with you favorites program or manually. Depending of your strategy, you will need to merge your current model with the DNA one and continue to refine. HTH Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 20/03/2018 19:28, Madhu Sudhan wrote: Dear all, I have an X-ray diffraction data of a protein-DNA complex at 2.3A resolution. I refined the structure, modeled the protein and the R-free reached to 0.35. I am observing extra density for the bound DNA molecule. As I am solving a protein-DNA complex structure for the first time, 1) Could anyone suggest me whether any automated program available to fit the DNA molecule into the density. 2) Also suggest me the other easiest ways, if any, to fit a DNA molecule into the electron density map. Thanks and Regards, Madhusudhan
Re: [ccp4bb] How to define the DNA bond between P and O3’ from two different symmetric units?
Dear Peng, to me your problem sound a bit strange, except if it's a palindromic sequence. I don't understand how you can have one part of the DNA in one asymmetric unit and one in another one. My question are : maybe you considering a NCS as a true symmetry and underestimate the unit-cell dimensions? Or you DNA has been degraded by DNase and the current size is not 20pb, in this case you are not supposed to create an artificial connection. Is the resolution good enough to be certain of the sequence ? Sorry, I don't provide any answer, but I am curious and try to understand what is going on. Hope this finally help. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 29/01/2018 20:28, Peng wrote: Hello, everyone， Recently, we solve a protein-DNA complex. 20bp-DNA was used for crystallization, but only 6bp was found in one symmetric unit. My question is： How to define the DNA bond between P and O3’ from two different symmetric units during my refinement? Peng
Re: [ccp4bb] CCP4i2 Error in wrapper
Hello, I am not sure but I had the same kind of problem using ccp4 on Linux "exotic" distribution which use python 3 by default. Check that your Linux installation has python 2.x as default python in your environement. Hope this guide a bit the trouble shooting. Nicolas Horrell, Sam a écrit >Hello CCP4bb, > >I'm having some trouble with my installation of CCP4i2. I keep getting a >persistent error about the "Wrapper" in various programs and am not sure what >I need to do to fix it. I have tried reinstalling via the package manager (64b >linux) and still get the error. So far it has happened in Molrep, Phaser, >Refmac and Morda (see below). I can provide log files if anyone thinks they >can help. > >Cheers, > >Sam > >MORDA > >-ERROR- morda_i2_gui:9 Error in wrapper morda_i2 0.1:: Failed starting >external process - is this program installed/accessible? >Process: /home/horrells/ccp4-7.0/libexec/python2.7 > >-ERROR- morda_i2_gui:47 Error in wrapper morda_i2 0.1:: Error in checking >external process after completion >exit status and code: 1 1 > >REFMAC > >-ERROR- None:201 Error in wrapper refmac 0.0:: Refmac returned with non zero >status >Exit code: 1 > >MOLREP > >-ERROR- None:9 Error in wrapper refmac 0.0:: Failed starting external process >- is this program installed/accessible? >Process: /home/horrells/ccp4-7.0/bin/refmac5 > >-ERROR- None:47 Error in wrapper refmac 0.0:: Error in checking external >process after completion >exit status and code: 1 1 > >PHASER (Expert and Basic) > >-ERROR- phaser_MR_AUTO_gui:48 Error in wrapper phaser_MR_AUTO 0.0:: No >description available >-ERROR- phaser_pipeline_gui:207 Error in wrapper phaser_pipeline 0.0:: No >description available >No output files in list >
Re: [ccp4bb] Weak density of RNA in a complex structure
Dear Chen, I will answer with some question : - How is the refinement going ? Is the RNA properly taking in count ? Which soft do you use ? - How do you solve the structure ? MR ? If it's MR did you use a model which contain both protein and RNA ? Did you try to solve with the protein only ? Maybe you are "forging" RNA. - Depending of your data (wavelenght and redundancy) you can try to calculate an anomalous difference map to see the phosphorus of the RNA to be more confident. - Last point, it could be the occupancy connected with the stability of the complex, maybe sometimes RNA is here sometimes not. I see that one time with two complex in the same ASU, one has one missing partner. For my side, I really like to refine my DNA-protein complex with Buster from global Phasing. It gives you a very nice and informative density map. If you have this possibility let's try. Hope to help. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 21/06/2017 09:34, Chen WeiFei wrote: Dear All, We have get a complex crystal and the resolution can be refined to nearly 1.84 Å. But the electron density of the RNA is very weak. In some datasets we can't find any density of the RNA and in other datasets we can see more or less some RNA density. For now we can build 6-7 nucleotides but we can't distinguish the rigth sequence. If anyone has the same problem and how to solve this problem. Best Regards, Dr Wei-Fei Chen College of Life Sciences, Northwest A University
Re: [ccp4bb] Protein or DNA crystals
Dear Joseph, I think, you already did the most classical test to determine what is in your crystal. Maybe you can try a destructive approach by washing the crystal prior to put them in Agarose gel do a short run and color with ethydium bromide. Simpler, you can also melt the crystal after washing them and do some spectroscopic experiment (measurement at 260 and 280 nm) the ratio will help ou to know what is in your crystal. I can add that I frequently obtained crystal of protein-DNA complex with this type of crystal growing condition (based on relatively small PEG). Sometimes it was only DNA. And most of the times when it was DNA only, crystal have a pentagon shape, like certain one on your image. If you have the opportunity to test diffraction, even at low resolution to obtain some information (cell params, matthew coeff) it will help you do decide what to do. And maybe are they already good enough to do some in-situ data collection or serial synchrotron experiment. Hope to help Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 19/06/2017 16:20, Joseph Ho wrote: Dear all: I would like to seek your opinion on our crystal hits. We are working on protein/dsDNA complex. By changing different protein and DNA (14-22bp) constructs, we recently got some hits from commercial screens using sitting drop vapor diffusion (very small xtals). The precipitant is PEG and the picture of crystals are attached. In this particular condition, it is 30%PEG3350, sodium succinate pH5.5 and 100mM NaCl. The crystal seems floating and sit in the bottom. We do some test shot from other conditions and it is not salt crystals. The crystals can suck in izit dye. I do some google and it seems izit dye also turns dsDNA crystal into blue. We also do UV/Vis microscope but no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp. This is our first time to work on protein/DNA complex crystals and we are not certain if this is just DNA or protein/DNA crystals. Can you provide your comments on our hits? Thank you for your help Joseph
Re: [ccp4bb] Large number of outliers in the dataset
Dear Juliana, all the statistics presented here looks good in terms of resolution cut (maybe I will be less sever). For me the point is about the mosaicity you report 1.90 it's high in my opinion. How looks you images? I am wondering if the indexation is really right. And maybe the complain of Xtriage about outlier is due to this high mosaicity. What is the diagnostic of Xtriage in terms of possible twinning? I am also wondering about a pseudo translation. Maybe try to re-processed your data in this direction. Hope to help. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 29/03/2017 17:56, Mark J van Raaij wrote: To be really convinced I think you should also compare the maps at 2.6 and 2.3 Å. If the 2.3 Å map looks better, go for it. If it doesn’t look better, perhaps you are adding noise, but the I/sigma and CC1/2 values suggest you aren’t. Perhaps try 2.5 and 2.4 Å also. And perhaps remove a well-ordered aa from the input model, refine at different resolutions and compare the difference maps for that aa. Or calculate omit maps at different resolutions and compare those. Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser.cnb.csic.es/~mjvanraaij <http://wwwuser.cnb.csic.es/%7Emjvanraaij> On 29 Mar 2017, at 17:44, Phil Evans <p...@mrc-lmb.cam.ac.uk <mailto:p...@mrc-lmb.cam.ac.uk>> wrote: It is not clear to me why you believe that cutting the resolution of the data would improve your model (which after all is the aim of refinement). At the edge CC(1/2) and I/sigI are perfectly respectable, and there doesn’t seem to be anything wrong with the Wilson plot. Th R-factor will of course be higher if you include more weak data, but minimising R is _not_ the aim of refinement. You should keep all the data I don’t know what xtriage means by “large number of outliers”: perhaps someone else can explain Phil On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira <juliana.olive...@lnbio.cnpem.br <mailto:juliana.olive...@lnbio.cnpem.br>> wrote: Hello, I have one dataset at 2.3 Å (probably it can be better, I/σ = 2.1 and CC1/2 = 0.779, the summary data is below), but when I perform Xtriage analysis it says that “There are a large number of outliers in the data”. The space group is P212121. When I refine the MR solution the Rfree stops around 30% and it doesn´t decrease (in fact if I continue refining it starts to increase). The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å: So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify about outliers anymore. I could refine the MR solution very well, the final Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a good structure. I run Zanuda to confirm the space group and it says that the space group assignment seems to be correct. Do you think that I can improve my structure and solve it at 2.3 Å or better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to justify the resolution cut, right? What should I say? Thank you for your help! Regards, Juliana Summary data: OverallInnerShell OuterShell Low resolution limit 51.51 51.51 2.42 High resolution limit 2.30 7.272.30 Rmerge 0.147 0.054 0.487 Rmerge in top intensity bin0.080 - - Rmeas (within I+/I-) 0.155 0.057 0.516 Rmeas (all I+ & I-)0.155 0.057 0.516 Rpim (within I+/I-)0.048 0.017 0.164 Rpim (all I+ & I-) 0.048 0.017 0.164 Fractional partial bias-0.006 -0.003 0.146 Total number of observations83988 2907 11885 Total number unique 8145307 1167 Mean((I)/sd(I)) 9.3 23.9 2.1 Mn(I) half-set correlation CC(1/2)0.991 0.998 0.779 Completeness 99.9 99.5 100.0 Multiplicity10.3 9.5 10.2 Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00 Space group: P212121 Average mosaicity: 1.90 Juliana Ferreira de Oliveira Brazilian Laboratory of Biosciences
Re: [ccp4bb] surface area
Hi Jiri, The surface are characterized by the probe you use to define them. Here it's water molecule I think. In such situation : Buried Surface Area are the surfaces */NOT/* accessible to the probe (water molec) it other word it doesn't imply that these BSA are contact surface between your proteins. Buried surface area = surface not accessible to the probe (because a groove too narrow for example) + interface (between two monomers) If you discussed about the contact between to monomer in my opinion the surface to focus is the interface surface not the whole BSA. But it's still possible to discuss about this point of view because interaction between two molecules could have remote effect (far from the interface) and for example modify the accessibility for the solvent in a different area. It really depend of what you are looking for : energy point of view, surface only... and if you consider to discuss small variations the resolution of the data and the reliability of your model may have important influence. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 13/03/2017 21:32, chemocev marker wrote: Hi I am comparing some protein complex with PISA analysis. Can someone make a note what is difference between interface area and buried surface area. I think buried surface area also include the interface area + the area encloses with in the protein. Does it make sense to mentioned the interface area separately if we count the buried area?? best Jiri
Re: [ccp4bb] Off-topic question about SEC
Dear Reza, in the past I had work with protein able to oligomerize reversibly but when oligomerization happened, even if I was able to separate and obtain monomeric protein, protein was not in a good condition. Have you try to characterize the two different states by DLS ? To discriminate "interaction with resin" that you suspect from oligomerization. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 12/01/2017 01:50, Christopher Colbert wrote: What's your monomeric molecular weight? Increased salt concentration can easily drive oligomerization. What is your evidence that it interacts with the resin? Cheers, Chris -- Christopher L. Colbert, Ph.D. Associate Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Reza Khayat <rkha...@ccny.cuny.edu <mailto:rkha...@ccny.cuny.edu>> Reply-To: Reza Khayat <rkha...@ccny.cuny.edu <mailto:rkha...@ccny.cuny.edu>> Date: Wednesday, January 11, 2017 6:42 PM To: "CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>" <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] Off-topic question about SEC All these make sense. Protein is very strange cause it goes from 60kDa (globular) to an apparent 360kDa. Process is reversible too. Reza Khayat, PhD Assistant Professor City College of New York Department of Chemistry New York, NY 10031 *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Keller, Jacob <kell...@janelia.hhmi.org <mailto:kell...@janelia.hhmi.org>> *Sent:* Wednesday, January 11, 2017 7:39 PM *To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> *Subject:* Re: [ccp4bb] Off-topic question about SEC Yes if it either A) oligomerizes B) significantly changes shape C) aggregates reversibly On option B: Lower NaCl could make the protein “appear” bigger by unfolding it a bit; hydrophobic interactions should be weaker in lower NaCl. JPK Artem www.harkerbio.com <http://www.harkerbio.com> "where wild SEC columns roam free" On Jan 11, 2017 7:22 PM, "Reza Khayat" <rkha...@ccny.cuny.edu <mailto:rkha...@ccny.cuny.edu>> wrote: Hi, Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes well within the resolution limits of the SEC with a symmetric gaussian A280 profile. I know that at lower [NaCl] the protein can elute later because it may interact with the matrix. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York Department of Chemistry New York, NY 10031
Re: [ccp4bb] Slightly OT: crystallization teaching resources for kids
Dear Evette, If I was is your situation (explaining nucleation and other concept). I will discuss in terms of energy. I mean obtaining the initial nuclei is the "costly" step in terms of energy. To represent that, out the classical curve of energy, I will use a metaphoric representation such as jump over a barrier and run after. With this analogy, it's possible to explain that the first step is difficult and the second more accessible. If the barrier is to high, it's impossible to continue and run. If you don't have any barrier it's easy to run and if you only have a small barrier is not to difficult to jump over and run. But It also allow you to explain that if you facilitate the apparition of the first "surface" thanks to appropriate method (seeding, dust...) you can help the first step (to continue with the barrier story, it like you have ladder to help, or the ability to decrease the size of the barrier. For why the crystal and how, I will maybe use the example of orange pyramid in the food store. Orange are stable together because they have enough contact, because they have relatively homogeneous shape. If you mixed orange with water melon it's difficult to obtain nice pyramid. For crystallization experiment which work, I have no Idea out of the one you already mentioned. Hope this help. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 30/12/2016 11:06, Radisky, Evette S., Ph.D. wrote: Can anyone point to some especially useful resources to help explain to kids (pre-chemistry, ~age 10-12) how and why molecules crystallize? Maybe a good online movie or animation? I am especially needing help with the concept of nucleation, and why nucleation is slower and then crystal growth faster once nuclei have formed. I have been supervising some experiments growing sucrose crystals from supersaturated solutions, which have worked really well, but I am having more difficulty in explaining the underlying fundamental concepts in a way that is understandable to the kids. Thanks! Evette Evette Radisky, PhD Associate Professor of Cancer Biology Mayo Clinic Cancer Center Griffin Cancer Research Building 4500 San Pablo Road Jacksonville, FL 32224 tel: 904-953-6372 fax: 904-953-0277
Re: [ccp4bb] Ubuntu Mate for Pymol and Coot 3D
Dear Paul, I am currently not working under Ubuntu OS, so I can't try your fix. But if my memory is still good, it was exactly the problem (the dynamic menus). And I am sure that some ccp4bb reader will be happy to find and try this solution. Thank you. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 20/12/2016 17:26, Paul Emsley wrote: On 20/12/16 16:21, Nicolas FOOS wrote: Dear Lei, I already try : Ubuntu Mate (no problem), Xubuntu (no problem), Ubuntu (standard) (I had problem with coot due to unity desktop). A shot in the dark, but maybe your problem is with dynamic menus? If so, try this: $ export UBUNTU_MENUPROXY=0 before running coot. Paul
Re: [ccp4bb] Ubuntu Mate for Pymol and Coot 3D
Dear Lei, I already try : Ubuntu Mate (no problem), Xubuntu (no problem), Ubuntu (standard) (I had problem with coot due to unity desktop). Also alternatively : Mageia5 (no problem), Manjaro (python3 as default in environment create some difficulty for several Macromolecular X-ray soft). In my opinion your choice is one of the more reliable. Most of the time the problem doesn't come form the OS, but more from the interaction between the OS and your graphic-card. And in your case that's precisely what is critical (since you want 3D in a good condition). Ubuntu works well with numbers of hardware configuration especially if you activate the proprietary repository in the packet manager. I used for a long time Xubuntu without any issues and was very happy with it. CCP4 and other soft are perfectly packed for this OS. HTH Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 20/12/2016 01:05, Xiao Lei wrote: Hi All, I am asking if anyone used Ubuntu Mate operating system and is this system good for Pymol and Coot 3D?
Re: [ccp4bb] intrasubunit rotation axis
Hi, not certain to understand the question. In chimera, you can create axis (for example in alpha helix) or directly draw one and do the angle measurement between differents axis. I imagine that you can create the "same axis" in both of your monomer and measure the angle between these axis. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 17/11/2016 23:48, Paul Emsley wrote: On 18/11/2016 03:57, chemocev marker wrote: Hi All I am interested to measure the symmetry axis of individual sub-unit (chain A & chain B) along with the symmetry axis of the heterdimer (AB). Each chain is also 2 fold axis, and I can measure by removing 1 chain and measure for the other and then combine all the symmetry axis in the 1 PDB file. Is there is a way to do it with out removing the either chain. I'm not sure that I fully understood the question, but you could try Coot's superpose_with_atom_selection, where you can specify the residue selection for the superposition e.g. Calculate -> Scripting -> Python superpose_with_atom_selection(0, 0, '//A/1-200', '//A/201-400', 1) will produce output like this: |0.9705, -0.0469, -0.2365| |0.2359,0.3887,0.8907| | 0.05015, -0.9202,0.3883| ( 37.8,-17.18, 13.83) Rotation - polar (omega,phi,kappa) 81.2326 -171.0041 68.0547 Rotation - euler (alpha,beta,gamma) 104.8719 67.1530 -93.1198 Translation - Angstroms 37.8021 -17.1809 13.8283 INFO: core rmsd achieved: 0.4517 Angstroems number of residues in reference structure: 200 number of residues in moving structure:200 number of residues in aligned sections (reference): 200 number of residues in aligned sections (moving): 200 number of aligned residues: 200 Paul.
Re: [ccp4bb] metal electron density detection
Dear Ansuman, this could help you, it explain how it's working on Phenix.refine Echols, N., Morshed, N., Afonine, P.V., McCoy, A.J., Miller, M.D., Read, R.J., Richardson, J.S., Terwilliger, T.C., and Adams, P.D. (2014). Automated identification of elemental ions in macromolecular crystal structures. Acta Crystallographica D /70/, 1104–1114. Nicolas Nicolas Foos PhD Structural Biology Group European Synchrotron Radiation Facility (E.S.R.F) 71, avenue des Martyrs CS 40220 38043 GRENOBLE Cedex 9 +33 (0)6 76 88 14 87 +33 (0)4 76 88 45 19 On 25/10/2016 11:04, ansuman biswas wrote: Hi all, Is there any program (like ARP/wARP) available to locate the correct metal atom automatically in the electron density ? best, Ansuman
Re: [ccp4bb] DNA interaction 2D plot software
Hello, you can try Nucplot. http://www.ebi.ac.uk/thornton-srv/software/NUCPLOT/ Very usefull and tunnable to find and show dsDNA/protein complex. Hope to help. Nicolas Le 16/10/13 21:23, Eike Schulz a écrit : Hello everyone, I would like to display the interactions of a protein dsDNA complex in a simplified 2D plot, similar to what LIGPLOT does for protein ligand interactions. In many articles you find interactions displayed in such a way but as far as I know those are hand-made. In my experience LIGPLOT itself is suboptimal if there are too many interactions to display … Thanks a lot in advance for your suggestions. Eike
Re: [ccp4bb] x-ray diffraction data analysis (XDS)
Dear Yu, in the CORRECT.LP, The I/Sig is very low for the resolution higher than 4.17 . If i were you i probably cut the resolution around 4 Å not 2.8 Å. Because of the I/sig and CC. This explain probably the bad electron density. Because, you have not strong information for the resolution higher than 4 Å. Hope to help Nicolas Le 30/08/13 10:59, Yu Longjiang a écrit : Dear all, I recently collected a dataset of a membrane protein. I first processed the data with HKL2000 to 2.8A ( the diffraction limit) at the beamline, and then treated the data again with XDS, although I am not familiar with this program. the quality of this data seemed not good compared with the other data we have collected before , and the electron density was worse after molecular replacement and refinement. the log file of HKL2000 is too large, so I just paste last statistics as below, the CORRECT.LP is also attached. Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 7.59 526.8 6.8 4.4 2.442 0.047 0.048 7.59 6.0354.4 2.5 2.4 1.727 0.112 0.104 6.03 5.2726.6 3.5 3.5 1.289 0.197 0.183 5.27 4.7927.4 4.9 4.9 1.201 0.231 0.228 4.79 4.4428.2 6.6 6.6 1.083 0.265 0.282 4.44 4.1828.2 8.0 8.0 0.939 0.304 0.357 4.18 3.9728.1 9.3 9.3 0.835 0.343 0.454 3.97 3.8028.610.410.4 0.802 0.366 0.501 3.80 3.6529.911.111.1 0.815 0.374 0.529 3.65 3.5330.011.711.6 0.824 0.384 0.550 3.53 3.4230.212.112.0 0.812 0.386 0.557 3.42 3.3229.812.212.2 0.817 0.389 0.565 3.32 3.2329.312.312.3 0.797 0.387 0.558 3.23 3.1528.412.312.3 0.802 0.387 0.559 3.15 3.0827.512.212.2 0.777 0.383 0.556 3.08 3.0226.712.012.0 0.762 0.383 0.551 3.02 2.9625.611.811.7 0.773 0.387 0.562 2.96 2.9025.111.711.6 0.734 0.379 0.543 2.90 2.8524.011.511.5 0.731 0.384 0.548 2.85 2.8023.711.511.5 0.754 0.383 0.551 All reflections 56.0 9.5 9.4 1.364 0.100 0.056 Any suggestion and comment about this dataset is greatly appreciated. Thanks a lot! YU
Re: [ccp4bb] Spots not getting indexed properly in protein-DNA complex
Dear Appu, one direction of your cristal has very anistropic diffraction. I think your cristal has grown like multi-layer. The problem is probably due to the DNA. If the different layer of your cristal are not very well aligned, you have this type of problem. In my opinion, you can try to select a subset of your data in the best direction and try to find your cell parameters and space grp. After that, if you have high symmetry parameters it could be sufficient with the subset, or you can try to reindex the other part of your data. I collect a lot of this type of data set. And all the time it was difficult to deal with. If you have time, and if your cristal has not suffer from radiation damage, you can try smaller oscillation angle. Hope to help you Le 01/08/13 15:44, Appu kumar a écrit : Dear all ccp4 user, We have collected a data set of 370 frames with 0.5 OSC for a protein-DNA complex on RAXIS IV detector. The spots look mostly clustered from 50A to 6A region but the whole data is completely spreaded to 2.8A. There are many spots which are very near to each other as if they were merged but closely placed. When we are trying to index the data, its not picking all the spots correctly and giving a unit cell dimension variable from 150 to 500A in one of the axis. Rest two axis axes of unit cell are almost similar ~ 55A, 111A. We tried processing with HKL2000 but not able to index it., I am attaching four 4 images for every 90 degree frames collected. Please look at these images and give your valuable input regarding indexing problem. your suggestions and support will be highly appreciated. Please guide me and help me sorting out the problem. Thank you
Re: [ccp4bb] Dose anyone see this ligand before?
Dear wei, i think you have to keep in mind what do you have in your cristal growing condition, and in you protein buffer. Because maybe you can find which molecules this ligand could be. It's difficult to help more, because we don't know you level of sigma for the map and the resolution of your dataset. But in all case, if you have an idea of what it could be, and you don't find restraint and molecules in existing library, you can use monomer library sketcher in ccp4 to sketch manually your molecule, and generate library of restraints. Hope to help you. Nicolas Le 16/07/13 17:56, Oganesyan, Vaheh a écrit : It actually looks much like pyrophosphate! If your protein is phosphatase and the extra density is in vicinity of the active site it might be the remaining product of reaction. /*/Vaheh/*/ *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Bosch, Juergen *Sent:* Tuesday, July 16, 2013 11:53 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Dose anyone see this ligand before? Special position ? sulfate-size ? Jürgen On Jul 16, 2013, at 11:35 AM, Wei Feng wrote: Dear all, I found some redundant density in my structure beween two molecule. (see picture 1 and 2) But I am not sure which ligand will be. Dose everyone see this ligand before? If so, can you tell the PDB code or send me the struture file? Thank you for your time! Wei ** picture1.jpgpicture2.jpg .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu 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.
Re: [ccp4bb] Diffraction image viewer with display of resolution circles
Hi Rafal, have you try ADXV ? I never try to use idiffdisplay, but i know that ADXV can display resolution circles. If your software is able to open the image, but can't show the circles. I think it's not really a problem with the image format, but with the information in the Header of your image. Normally you can find information about the resolution, and detector position etc... If you detector control software didn't writte a complet Header, or if your image viewer is not able to read the Header it could be the source of your problem. Hope to help you. Nicolas Le 23/05/13 10:16, Rafal Dolot a écrit : Dear CCP4 users, I'm looking for the diffraction image viewer, which will be able to display of resolution circles and export it to new image. I tried use idiffdisp, but after choose of the Show/clear resolution circles, there is no action. Images were collected using Rayonics MX-225 detector - maybe detector format is a problem? Best regards, Rafal |--| |Rafal Dolot, Ph.D.| | | |Polish Academy of Sciences| |Centre of Molecular and Macromolecular Studies| |Department of Bioorganic Chemistry| |Sienkiewicza 112 | |90-363 Lodz, Poland | |Phone: +48(42)6803215 | |Cell: +48 502897781 | |--| -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] fplc system
Dear Alex, i work with Purifier. To my mind, this is a very nice system. High reliability. We are a lot of differents users and we share the same system, some of us are not very implicated in the cleaning and maintenance procedure, nonetheless the Akta works well. Akta Purifier with 3 wavelenghts is very usefull. For the best usage, you have to use it in cold room, or with a good cold cabinet. I saw one time the Akta Avant (for testing), it looks like a ferrari (shining red color for the exterior shell ;-) ), this product propose you to prepare Buffer with stock solution. Your sample after collection, is keeping in cold compartment. This product have a lot of very usefull and high-tech specs. But i think the good question is : what i want do with my FPLC Because, the cost is very different, and if i have to choice, for my usage, i prefer to buy Purifier, with good fractionation system, and some good column instead of the ferrari. The major interest with the Avant, is that this system has the capacity to work, at high speed (fast flow) like the Akta Express, and the other side, you can set very advanced parameters, to prepare your buffer. Nothing else to my knowledge can prepare the same variety of buffer automatically. And finally, you can develop very fine and advanced purification protocol. Hope to help you. Nicolas Le 28/03/13 20:39, Alexandra Deaconescu a écrit : Dear crystallography enthusiasts: We are shopping for a new fplc system. While have pretty much decided to buy a GE Healthcare (Akta) rather than a Biorad system, we are interested in finding out your opinions about the new models that are currently on the market, such as the Akta Avant and the Akta Pure, and how they compare to the Purifier. Any comments would be much appreciated! Bests, Alex
Re: [ccp4bb] Build DNA to fit density
Hi Wei, If you have an interpretable density, the efficientest way in my opinion, is to construct manually (only 6bp). You can use coot and the button add residue. Coot can add nucleotide directly at the extremity of your existing nucleic acid model. Hope to help you. Nicolas Le 18/03/13 03:43, Wei Shi a écrit : Hi all, I am refining a structure of protein-DNA complex with coot. The DNA in my search model is shorter than the DNA in the crystal, and now I could see the density for extra DNA(6 base pairs) on either end of the search model DNA. But, I don't know how to build the extra DNA back to fit the density or whether I should build the whole DNA manually to fit the density. I used calculate- other model tools- ideal DNA/RNA to generate a 6 base pairs (B form) and then, I use calculate- model/fit/refine-rotate/Translate molecule to move the 6 bp long stretch of double strand DNA to fit the density, but it's hard for me to fit the DNA into the density and it seems that the B form DNA I generate doesn't fit the density well. I am wondering how to fit the DNA into the density and whether we could fit the DNA into density like we add amino acid to fit the density. Thank you so much! Best, Wei
Re: [ccp4bb] RNA 3D structure alignment
Dear Chen, you can find lsqkab and Topp in ccp4 : coordinates utility and superpose molecule (if you use the GUI). Hope to help you. Nicolas Le 14/03/13 21:53, Chen Zhao a écrit : Dear all, I am now struggling to align two 3D RNA structures. I know there are a bunch of web servers, but they either just generated a pdb file with a single aligned structure, or they left the ligand out. Does any of you have some recommendations? Alternatively, is there some software that can calculate the transformation matrix between the coordinates in 2 pdb files? Then I could add the ligand back by myself. I suspect that Matlab is able to do this, but I would save it as the last resort. Thank you so much! Best, Chen
Re: [ccp4bb] Assemble Protein-DNA complex
Dear Wei, If i understand your different experiment, you try to obtain your protein DNA complex at different salt concentration with different method to reach the final concentration. I read that you try 150 mM KCl + 150 mM NaCl as high concentration salt, it result in 300 mM cations and 300 mM Cl. To my mind, and according your experiment it's too high. But when you try low concentration you dialized the protein alone in 30 and 100 mM salt concentration. In my opinion the best way, is to dialize the protein mixed with the DNA. Because the protein will probably be stablize by DNA. If the protein meet slowly DNA at the same time the salt concentration decrease, you minimize the precipitation (it's your third experiment in fact). You have not precised the final salt concentration with this method. If you try 30 mM, maybe it's too low, i suggest 100mM Salt. I remember paper about nucleosome where author explain solution with multiples steps dializis. It's more gradual and it could help if you want reach very low salt concentration. One other idea, is to play with the pH, because of the protein's pI. In function of the pI you have to try different pH for the complex preraration. You can optimise the protein DNA interaction through the protein charge. HtH Nicolas Le 09/11/12 17:14, Wei Huang a écrit : Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] Fwd: [ccp4bb] RE : [ccp4bb] Strange diffraction image
Dear Chang, i have seen ATP diffraction, it's not very different of your image. Maybe you have only ATP in your crystals? Best regard Nicolas Le 12/10/12 14:56, Chang Qing a écrit : Dear Tim I think your explanation is logical. But I tried ADP as ligand first and got crystals and diffraction. ATP in additive kit was found to improve the quality of crystals from cluster to single crystal. CsCl can go on improving the quality and finally I got crystals like this. Is it possible that I get some strange crystals such as CsMgCl3 or something else? Best regard Chang -- Forwarded message -- From: Tim Gruene t...@shelx.uni-ac.gwdg.de Date: 2012/10/12 Subject: Re: [ccp4bb] RE : [ccp4bb] Strange diffraction image To: Chang Qing robie0...@gmail.com 抄送： CCP4BB@jiscmail.ac.uk -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Chang, What makes you think spots from salt crystals should not be as large? You have got an ordinary small molecule crystal (or probably a cluster of them) in the beam, with a unit cell somewhat bigger than that of ice judging by the extra real ice rings you've got. Hydrolysis of ATP and Mg2+ present - it's probably (Mg)3(PO4)2- it is very unsoluble http://en.wikipedia.org/wiki/Solubility_table. Best, Tim On 10/12/2012 09:29 AM, Chang Qing wrote: Hi, Thanks for answering my question. I think I'd better provide more informations. These four images were taken from one crystals. The distance of image1-2 is 250mm, and image3-4 is 100mm. I checked more than 10 crystals and results were similar. The spots look very large. The lowest resolution is about 6A. As my protein can hydrolyze ATP, so protein buffer with 5mM of ATP. There is 0.2M of MgCl2 in precipitant buffer with PH7.0. 0.06M of CsCl can improve quality of crystals. I also setup control, in which target protein was not added, and could get nothing in it. I don't think it is a protein crystal. But salt spot should not be so large. And the rings are not just ice-ring. As I got crystal first in hampton crystal screen kit with MgCl2, TrisHCl and PEG4,000, there are only rings in images from 5-6A to about 3A. 2012/10/12 THOMPSON Andrew andrew.thomp...@synchrotron-soleil.fr: Hi Chang No mention of the resolution limit / oscillation range (I think I can see an ice ring, so I would guess 2.5 A?), but it looks like salt to me, with some weaker satellite peaks that may be something weird like an incommensurate phase. Did you try to index? Cheers Andy De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Chang Qing [robie0...@gmail.com] Date d'envoi : vendredi 12 octobre 2012 08:11 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Strange diffraction image Hi, everyone: I just got some strange diffraction images from crystals with triangular pyramid shape. I think this should not be protein diffraction. I never saw so strange images like this. Does anyone know what it is? Is it a kind of salt diffraction? Thank you very much Chang - -- - -- 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 Mozilla - http://enigmail.mozdev.org/ iD8DBQFQeAheUxlJ7aRr7hoRAibDAJ9yAfiSwNmh8R4tGwUIwFEZno2qWACfStCM y+xKb+FGGglmv8lTL9Ej8ZQ= =ub/P -END PGP SIGNATURE-
Re: [ccp4bb] how to overlap DNA
Dear Leo, I use *superpose* included in the ccp4 programms suite. You can manage a lot of parameters. In my case for exemple, i use the superpose specified atoms/residues I defined the model which move et the one wich is the reference. I precise the residues range (in DNA case residue means you base). And it work very fine. But your own method depend of what you want to do with your overlaped structure. Hope to help you. Nicolas Le 03/05/12 03:18, Junfeng Liu a écrit : Dear All, Does anybody has the experience to overlap DNA in the protein complex? I have tried the LSQ in Coot and it works ok but not perfectly. Could you please tell me a better way to do that?Or can Coot be used to overlap DNA/RNA on their back bone by LSQ or SSM ? At the same time, are there some software or servers can be used to search the homology DNA in the database like the Dali on protein? Thanks in advance! Best wishes leo
Re: [ccp4bb] X-PLOR
Hi, I find some information here : http://www.csb.yale.edu/userguides/datamanip/xplor/xplorman/htmlman.html And for a more usefull answer, they explain that : The hbuild statement tries to build the position of any selected hydrogen based on the position of the heavy atom antecedents (Brünger and Karplus 1988). It works in a general way and can be used with any empirical force field. It performs local energy minimization in cases where the placement of the hydrogens is not unique. Waters close to the macromolecule are placed first, followed by waters that are farther away. Several iterations can be carried out to reach self-consistency. The minimization uses the energy function (Eq. 4.1 http://www.csb.yale.edu/userguides/datamanip/xplor/xplorman/node113.html#eqetotal) except that the specification of constraints interaction statements (Section 4.7 http://www.csb.yale.edu/userguides/datamanip/xplor/xplorman/node131.html#constraintsinteraction) are ignored. The hydrogen building facility does not use the improper or dihedral information in the topology file (Section 3.1.1 http://www.csb.yale.edu/userguides/datamanip/xplor/xplorman/node46.html#topologystatement). The chirality of methyl and methylene groups is determined by the order of four substituents to the central carbon atom as specified in the molecular structure. By changing the order in which the atoms are defined in the residue statement (Section 3.1.1 http://www.csb.yale.edu/userguides/datamanip/xplor/xplorman/node46.html#topologystatement), the chirality of the center is changed. Hydrogens are recognized by X-PLOR by their mass. All atoms with a mass less than 3.5 amu are considered hydrogens. Certain simulated annealing protocols (e.g., Section 20.3.3 http://www.csb.yale.edu/userguides/datamanip/xplor/xplorman/node395.html#dgsaprotocol) make use of artifically increased masses. If hydrogen building is employed in these protocols, the hydrogen building routine will return an error statement. In this case the user has to reset the mass of the hydrogen atoms to their physical values. To save CPU time, it is suggested to carry out the hydrogen building without nonbonded energy terms. The coordinates then will need minimization to relieve possible bad contacts between the initial hydrogen placements. You can find more information about the CHARMM force field apllied to the Explicit polar hydrogens of Nucleics acids and proteins HTH Nicolas Le 27/04/12 15:34, Nadir T. Mrabet a écrit : Hi, Could someone explain to me the scientific details of the protocols used in X-PLOR to (1) build explicit hydrogen atoms onto X-ray structures and (2) optimize their positions? Many thanks in advance. Greetings, Nadir
Re: [ccp4bb] XSCALE!!! ERROR
Hello Bashir, Maybe it's because your dataset present too much weak diffraction spots. You can try to change the setting for the strong pixel detection. You can Try to decrease a little bit de I/sigma. Have you define the resolution range? I read on your message that you resolution start at 5 A. To my mind it's too high, you should have more low resolution. Perhaps your problem com from this specificity of you dataset. Try first to set your resolution range from inf. to 3.25 Hope to help you. Nicolas Le 07/09/11 21:39, Muhammed bashir Khan a écrit : Dear All; I am trying to to xsale several data sets together, but it gives an error of !!! ERROR !!! INSUFFICIENT NUMBER OF COMMON STRONG REFLECTIONS. PROGRAM IS UNABLE TO PRODUCE A SCALED DATA SET. resolution of data sets is ranged from 3.25 to 5A Any suggestion will be highly encouraged. Thanks in adv. Bashir
Re: [ccp4bb] coot torsion angle restraint
Hi, i think that this option for the real space refinement is for _all_ the torsion angle. You can find more informations with this link : http://www.ysbl.york.ac.uk/~emsley/coot/doc/chapters/user-manual_5.html#SEC92 In the 5.1 section. Hope to help you. Nicolas Le 24/07/11 02:24, zhang yu a écrit : Hi, I am confused by the torsion angle restraint for real space refinement in coot. Is the Torsion angel restraint for side chain or for main chain? Yu -- Yu Zhang HHMI associate Waksman Institute, Rutgers University 190 Frelinghuysen Rd. Piscataway, NJ, 08904 -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] hello
Hello Afshan, Maybe the programm that you use for refinement need specific entry with restraint for your modified amino acids. More precisely, i think about the *.cif file for exemple. HTH. Nicolas Le 21/07/11 17:13, Afshan Begum a écrit : Dear all, I have facing one problem during the refinement of my protein . Actually in my protein there are some modified amino acids are present like Cystein is modified into CME which i can get easily from monomer libraray in coot . but after refinement in Pdb text file indicated some gaps while in the structures there are no gap in between these amino acids so if any one suggest me what to do. I would appreciate your kind suggestions. LINKRGLU A 142 LEU A 144gap LINKRSER A 328 GLY A 330gap LINKRLEU A 138 GLU A 140gap LINKRGLU A 126 ASP A 130gap LINKRSER A 246 GLY A 248gap Many thanks for your time Best regards Afshan -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] DNA analysis with Curves+
Hello Edward,
I am not really certain for my explanation, but your error message about
the namelist could be provoked by a problem in your input files. In fact
you have to oriented each strand and define limits of the two strands.
If you do a mistake with the orientation you can have this type of
message. You can try different combination with this part of your
command line :
2 1 -1 0 0
1: 12
24:13
Maybe the key could be in this part of your command line atless for the
input error message.
To my mind you have to edit your input file because its format is not
adaptated for this programm. The format wich is too recent or with to
much informations are not well suported.
I hope i help you.
Sincerly . Nicolas
Le 03/02/11 21:05, Eddie Pryor a écrit :
Hi, All,
I am trying to analyze DNA from a recent structure with the program
Curves+ (R. Lavery et al, Nuc. Acids. Res (2009) 37:17 5917;
http://gbio-pbil.ibcp.fr/Curves_plus/Curves+.html). I have downloaded
the files, and untarred them to a directory curves on my machine.
Running the Make command on these files yielded no errors, and I have
the executable file Cur+ in this directory. I am trying to run this
program, first using the example data that is provided (the
Drew-Dickerson Dodecamer), but am having some difficulties. The
resource for Curves gives the following command to run the program:
/Users/RL/Code/Cur+ !
inp file=str/1bna, lis=r+bdna,
lib=/Users/RL/Code/standard,
end
2 1 -1 0 0
1: 12
24:13
!
On my machine, the directory structure is slightly different:
- the executable Cur+ is in a folder named curves
- the files standard_b.lib and standard_s.lib are also in this folder
- the pdb file 1bna.pdb is in the subfolder data
I have tried to run the program by doing the following:
[x@zeus ~/curves]$ Cur+ !
? inp file=data/1bna, lis=r+bdna,
? lib=standard, end
? 2 1 -1 0 0
? 1:12
? 24:13
? !
The question mark prompts appear upon hitting enter after each line.
The only error message I get when I try to run this is:
Error in namelist input for
I have even tried the following (entering all if the input files on
the same line):
[x@zeus ~/curves]$ Cur+ ! inp file=data/1bna, lis=r+bdna,
lib=standard, end
? 2 1 -1 0 0
? 1:12
? 24:13
? !
Which gives the following errors:
inp: Command not found
end: Not in while/foreach
At line 28 of file nml.f
Fortran runtime error: End of file
I am hoping that some of you have had success with running this
program and can offer any advice. I would really appreciate it!
Thanks!
Edward Pryor
Ph.D. Candidate
Tom Hollis Lab
Department of Biochemistry
Center for Structural Biology
Wake Forest University School of Medicine
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Re: [ccp4bb] SP Sep HP tight binding of proteins
Hi Meg, If the bigger peak is appearing when you wash with 1M NaOH, i think your protein is precipitated on the the column. If you have your protein relatively pure after the precedent step, maybe you can try different buffer. You can probably find a better buffer than this one. If your protein is in good condition before the column, maybe its a problem with the column type. Maybe you can try an other SP. If your protein have Cys, maybe you can try to add som DTT or 2-mercaptoethanol or something like this. In my opinion the best start is to detemined the better pH and after try some other stuff like reducing agent, light detergent... (triton X-100, Glycerol...). Nicolas Le 04/01/11 10:34, megha goyal a écrit : Dear All, We used SP sepharose high performance as second stage Ion exchange chromatography for polishing the product. We did get pure product but yield obtained was mere 25%. Our protein has a pI of 5.5 - 6.0 and we had used 25 mM Na Acetate buffer pH 4.5 for loading and same buffer with 1M NaCl for elution, 25 C.V. linear gradient. Can you suggest some changes that i can incorporate to increase the yield i.e additives to be added or some change in pH etc. I tried elution with arginine HCL as elution buffer as was recommended in one paper, but the yield obtained was even less. On washing with 2M NaCl ther is not much peak appearing but on washing with 1M NaOH substantial peak appears. Kindly help me through this. meg -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] DNA-protein complex.
Hi, maybe you solved your problem, if not, you can try this : You prepare your protein at high salt concentration. After that you add your ssDNA directly and you proceed by dialysis. You prepare your dialysis with a low salt concentration buffer. You can use different buffer by step in order to avoid a too fast salt concentration decrease. This method can allow progressive assembly of your complex. Nicolas Le 31/10/10 08:06, dengzq1987 a écrit : Hi all, I want to sovle the structrue of ssDNA -protein complex.but the protein are unstable at low salt concentration,so we use 1M Nacl in the buffer. the high salt content may disrupt the complex.and I don't know which ssDNA lenght try fist.any suggestion or experience are welcome. thank you! 2010-10-31 dengzq1987 -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Mutating bases in DNA
Hi Rex, If you have to mutate only few bases, maybe you can proceed manualy with coot. And maybe after mutate refine at less for geometricals parameters of your new base. Nicolas. Le 13/09/10 16:43, Rex Palmer a écrit : Does anyone know of a program that will mutate a given base in the pdb of a DNA structure? Many thanks in advance. Rex Palmer Birkbeck College, London -- This message has been scanned for viruses and dangerous content by *MailScanner* http://www.mailscanner.info/, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
