[ccp4bb] Phaser question
Hiyya all, I have a question about the latest Phaser output, concerning TFZ = and TFZ == . I do not know how to interpret outputs of the type TFZ = 5.2 TFZ == 54.1; TFZ = 5.8 TFZ == 63.0; TFZ = 6.4 TFZ == 19.7 (these are real TFZ figures coming from Phaser log files). I used to analyse the Phaser output using TFZ, when only a single number was given. Now with two figures to consider, I do not know what to think of it any more. Any ideas out there ? And best wishes for an enjoyable end-of-the-year season (be it in the cold or in the sun depending on which side of the planet you sit). Fred. -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494)
Re: [ccp4bb] Phaser question
Hi Fred, Send me the logfiles (off-line), because this shouldn't be happening and I'd like to have a look. That said, we've been seeing some similar problems in certain circumstances, i.e. B-factor refinement refines to significant negative B-factor values, and data at high resolution have very low signal-to-noise. If those are the circumstances, we're currently working on a fix and maybe we can get you to test it on your data once it's implemented. All the best, Randy On 5 Dec 2013, at 08:36, vellieux frederic.velli...@ibs.fr wrote: Hiyya all, I have a question about the latest Phaser output, concerning TFZ = and TFZ == . I do not know how to interpret outputs of the type TFZ = 5.2 TFZ == 54.1; TFZ = 5.8 TFZ == 63.0; TFZ = 6.4 TFZ == 19.7 (these are real TFZ figures coming from Phaser log files). I used to analyse the Phaser output using TFZ, when only a single number was given. Now with two figures to consider, I do not know what to think of it any more. Any ideas out there ? And best wishes for an enjoyable end-of-the-year season (be it in the cold or in the sun depending on which side of the planet you sit). Fred. -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494) -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 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
[ccp4bb] rotation peak number in new version phaser
hi all, I try to solve the crystal structures of a mult-domain protein with phaser. I cannot solve the structure by automatic search using domain structure as the models. I tried to do the rotation search using the fast or brute rotation function. I defined the rotation search peaks number is 1000. But I only get no more than 100 peaks. I tried the phaser in ccp4-6.3.0 and ccp4-6.4.0. But I can only get much less peaks than I required in the rotation. But the old version phaser can give about 1000 peaks if I use brute rotation function ask for 1000 peaks. How can I more rotation peaks in the new version phaser ? Thank you. Lisa
Re: [ccp4bb] small crystals
Dear Careina – Orthogonal crystallization methods can be of great utility when optimizing crystallization conditions, since they offer a different sampling of the crystallization space. Orthogonal methods include: liquid-liquid diffusion, microbatch and dialysis. Liquid-liquid diffusion is known to be a productive crystallization method and a recent paper from the Sundberg lab demonstrated that crystallization by liquid-liquid diffusion could improve both the size and diffraction quality of crystals of an endo-b-N-acetylglucosaminidase, EndoS, from Streptococcus pyogenes. The paper can be found here: http://scripts.iucr.org/cgi-bin/paper?nj5178 Description of the crystallization device can be found here: http://scripts.iucr.org/cgi-bin/paper?S1744309111024456 and http://www.microlytic.com/crystal-former Using microseed matrix screening could also improve your crystal quality. You can find information on the approach here: http://pubs.acs.org/doi/abs/10.1021/cg2001442?journalCode=cgdefu Best regards, Morten Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- TotalCare Message Security: Click below to verify authenticity http://www.exchangedefender.com/verify.asp?id=rB5EYXWs004083from=m...@microlytic.com
Re: [ccp4bb] small crystals
Hi, can you give a bit more information... Can you concentrate the protein easily to a higher concentration, let's say 2-3 times from what you have now, without precipitation? What is the buffer of your protein stock solution at the moment? At what temperature and what precipitant are you using? - Jeroen - showers of crystals Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. TotalCare Message Security: Check Authenticity -- Dr. Jeroen R. Mesters Deputy and Group Leader Institute of Biochemistry, University of Lübeck Ratzeburger Allee 160, 23538 Lübeck, Germany phone: +49-451-5004065 (secretariate 5004061) fax: +49-451-5004068 http://www.biochem.uni-luebeck.de http://www.iobcr.org -- If you can look into the seeds of time and tell which grain will grow and which will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act I, Scene 3) -- Disclaimer * This message contains confidential information and is intended only for the individual named. If you are not the named addressee you should not disseminate, distribute or copy this e-mail. Please notify the sender immediately by e-mail if you have received this e-mail by mistake and delete this e-mail from your system. * E-mail transmission cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. If verification is required please request a hard-copy version. Please send us by fax any message containing deadlines as incoming e-mails are not screened for response deadlines. * Employees of the Institute are expressly required not to make defamatory statements and not to infringe or authorize any infringement of copyright or any other legal right by email communications. Any such communication is contrary to Institute policy and outside the scope of the employment of the individual concerned. The Institute will not accept any liability in respect of such communication, and the employee responsible will be personally liable for any damages or other liability arising. Employees who receive such an email must notify their supervisor immediately. --
[ccp4bb] PhD opportunity
Dear colleagues, I am currently looking for up to two PhD students to work on proteins involved in double strand DNA repair. Details on the projects can be found here: http://www.findaphd.com/search/ProjectDetails.aspx?PJID=48449 http://www.findaphd.com/search/ProjectDetails.aspx?PJID=48456LID=456 The Eastbio project is open to candidates that meet the residency criteria, the other project is open to overseas students. Both are competition funded projects. Informal enquiries should be addressed to laura.spagn...@ed.ac.uk, the application is online: http://www.ed.ac.uk/schools-departments/biology/postgraduate/pgr/how-to-apply ***The closing date is December 16th*** Laura Laura Spagnolo ISMB University of Edinburgh laura.spagn...@ed.ac.uk http://spagnolo.bio.ed.ac.uk/ +44 (0)131 6507066 -- The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
Re: [ccp4bb] small crystals
Hi All On similar lines, I have been trying to optimize crystallization conditions for my protein. Initially, I had showers of needles in a PEG screen which did not really improve after screening around the condition. So, I seeded these needles into all the screens that I have available and I have plate like crystals which do not diffract at home in MPD (~15%), 0.1 M sodium acetate, 0.2 Mgcl2/cacl2 at 294 K. I tried incubating at 287 K, but that did not yield any useful results.The protein itself is in 50mM MOPS, 10% glycerol pH 7.5. I could try to take of glycerol but i cannot concentrate the protein more than ~ 5mg/ml which clearly was not sufficient to achieve crystallization. Any advice is deeply appreciated. Thank you cheers Mahesh On Thu, Dec 5, 2013 at 10:03 AM, mesters mest...@biochem.uni-luebeck.dewrote: Hi, can you give a bit more information... Can you concentrate the protein easily to a higher concentration, let's say 2-3 times from what you have now, without precipitation? What is the buffer of your protein stock solution at the moment? At what temperature and what precipitant are you using? - Jeroen - showers of crystals Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. *TotalCare* Message Security: Check Authenticityhttp://www.exchangedefender.com/verify.asp?id=rB5EYXWs004083from=m...@microlytic.com -- Dr. Jeroen R. Mesters Deputy and Group Leader Institute of Biochemistry, University of Lübeck Ratzeburger Allee 160, 23538 Lübeck, Germany phone: +49-451-5004065 (secretariate 5004061) fax: +49-451-5004068 http://www.biochem.uni-luebeck.de Http://www.biochem.uni-luebeck.de http://www.iobcr.org Http://www.iobcr.org -- If you can look into the seeds of time and tell which grain will grow and which will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act I, Scene 3) -- Disclaimer * This message contains confidential information and is intended only for the individual named. If you are not the named addressee you should not disseminate, distribute or copy this e-mail. Please notify the sender immediately by e-mail if you have received this e-mail by mistake and delete this e-mail from your system. * E-mail transmission cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. If verification is required please request a hard-copy version. Please send us by fax any message containing deadlines as incoming e-mails are not screened for response deadlines. * Employees of the Institute are expressly required not to make defamatory statements and not to infringe or authorize any infringement of copyright or any other legal right by email communications. Any such communication is contrary to Institute policy and outside the scope of the employment of the individual concerned. The Institute will not accept any liability in respect of such communication, and the employee responsible will be personally liable for any damages or other liability arising. Employees who receive such an email must notify their supervisor immediately. --
Re: [ccp4bb] small crystals
depending on how extensively you have screened so far, the most efficient thing to do may be to change the protein: different orthologs, truncations, mutagenesis of entropy rich clusters, change of tag location or tag cleavage etc. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mahesh Lingaraju [mxl1...@psu.edu] Sent: Thursday, December 05, 2013 5:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] small crystals Hi All On similar lines, I have been trying to optimize crystallization conditions for my protein. Initially, I had showers of needles in a PEG screen which did not really improve after screening around the condition. So, I seeded these needles into all the screens that I have available and I have plate like crystals which do not diffract at home in MPD (~15%), 0.1 M sodium acetate, 0.2 Mgcl2/cacl2 at 294 K. I tried incubating at 287 K, but that did not yield any useful results.The protein itself is in 50mM MOPS, 10% glycerol pH 7.5. I could try to take of glycerol but i cannot concentrate the protein more than ~ 5mg/ml which clearly was not sufficient to achieve crystallization. Any advice is deeply appreciated. Thank you cheers Mahesh On Thu, Dec 5, 2013 at 10:03 AM, mesters mest...@biochem.uni-luebeck.demailto:mest...@biochem.uni-luebeck.de wrote: Hi, can you give a bit more information... Can you concentrate the protein easily to a higher concentration, let's say 2-3 times from what you have now, without precipitation? What is the buffer of your protein stock solution at the moment? At what temperature and what precipitant are you using? - Jeroen - showers of crystals Original message Subject:[SURESPAM] [ccp4bb] small crystals From:Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com To:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Cc: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. TotalCare Message Security: Check Authenticityhttp://www.exchangedefender.com/verify.asp?id=rB5EYXWs004083from=m...@microlytic.com -- Dr. Jeroen R. Mesters Deputy and Group Leader Institute of Biochemistry, University of Lübeck Ratzeburger Allee 160, 23538 Lübeck, Germany phone: +49-451-5004065tel:%2B49-451-5004065 (secretariate 5004061) fax: +49-451-5004068tel:%2B49-451-5004068 http://www.biochem.uni-luebeck.deHttp://www.biochem.uni-luebeck.de http://www.iobcr.orgHttp://www.iobcr.org [X] -- If you can look into the seeds of time and tell which grain will grow and which will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act I, Scene 3) -- Disclaimer * This message contains confidential information and is intended only for the individual named. If you are not the named addressee you should not disseminate, distribute or copy this e-mail. Please notify the sender immediately by e-mail if you have received this e-mail by mistake and delete this e-mail from your system. * E-mail transmission cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. The sender therefore does not accept liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. If verification is required please request a hard-copy version. Please send us by fax any message containing deadlines as incoming e-mails are not screened for response deadlines. * Employees of the Institute are expressly required not to make defamatory statements and not to infringe or authorize any infringement of copyright or any other legal right by email communications. Any such communication is contrary to Institute policy and outside the scope of the employment of the individual concerned. The Institute will not accept any liability in respect of such communication, and the employee responsible will be personally liable for any damages or other liability arising. Employees who receive such an email must notify their supervisor immediately. --
Re: [ccp4bb] small crystals
Hi, sorry, it should read salt in not inverse salt in ... - J. - Am 05.12.13 19:55, schrieb mesters: Hi, Showers of crystals often occur if the protein is not that soluble/happy/stable in the solute. The solubility of a protein depends on its concentration, its pI, pH of solute, temperature, compounds in the solute (e.g. salts and small organic molecules), construct used, to name a few. So, to increase the solubility, you need to change one these parameters. Salts in general are said to inverse salt in the protein thus making it more soluble. Hofmeister published an interesting paper on this in 1888! NaCl for example is placed in the middle of the Hofmeister series, neutral so to speak, and practice shows it is often a good choice to start with at a concentration of 150-200 mM. However, occasionally it is not a good choice and will have negative effects. The choice of salt basically depends on the pI of the protein and the pH of the solute/crystallization condition. Have a serious look at the chapter by Madeleine Riess-Kautt about the Hofmeister series in A. Ducruix und R. Giegé book with title Crystallization of Nucleic Acids Proteins. A practical Approach (Oxford IRL-University Press (1992)). For a normal hen egg white protein (pI/IEP below the pH, protein overal negatively charged) he was working on, ammoniumsulfate will precipitate (stabilize/crystallize) the protein while a perchlorate salt (destabilize) will dissolve the protein completely at high concentrations. As you generally need high protein concentrations for starters in crystal growth, you thus need to add a salt that dissolves the protein slightly without denaturing it (low concentration 50 -200 mM in the solute). Then, you can add a second salt (on the opposite site of the series) or precipitant like PEG to crystallize it. However, for several proteins the pI is located above the pH of the solute. In these cases the protein is positively charged and the Hofmeister series turns around! We recently had a case like this producing lost of tiny plate-like crystals. Changing the protein concentration or the precipitant concentration, or hanging to sitting etc. did not help at all. We finally found out we had to add some ammoniumsulfate to solubilize/dissolve the protein first and got very nice single and large crystals in the end. In your case, try to replace the glycerol by the proper choice of salt! You can also try to exchange MOPS (zwitterion) for another buffer compound. All this will also change the crystallizaiton behaviour. You will most probably need to redo the screening... - J. -
[ccp4bb] positions available
Post Doctoral Positions available in Australia *Postdoctoral Research Fellows * ** *Department of Biochemistry and Molecular* *Biology, Faculty of Medicine, Nursing and Health Sciences* *The laboratory*: The Rossjohn laboratory, in a broad collaborative network that includes lead national and international researchers, has provided profound insight into T-cell biology, specifically defining the basis of key immune recognition events by T-cells.The laboratory has explained pre-T-cell receptor (TCR) self-association in T-cell development, and how the TCR specifically recognises polymorphic Human Leukocyte Antigen (HLA) molecules in the context of viral immunity and aberrant T-cell reactivity. Laboratory members have unearthed mechanisms of HLA polymorphism impacting on drug and food hypersensitivities, as well as Natural Killer cell receptor recognition. The laboratory has pioneered our understanding of lipid-based immunity by the innate Natural Killer T-cells (NKT) and the role of MAIT cells in recognizing vitamin B metabolites.Many researchers within the laboratory have secured national fellowships and awards. For more details on the research themes, fellowships awarded and publication outputs see: http://research.med.monash.edu.au/rossjohn/** *The role:* Prof. Jamie Rossjohn, NHMRC Australia Fellow, is seeking highly motivated and talented post-doctoral scientists to join his research group. The selected candidates will work on one of two projects, a) the investigation of NKT-CD1d recognition and b) HLA molecules in adaptive immunity. The applicant should hold a PhD in Biochemistry, or Molecular Biology or Structural Biology with an interest in Protein Chemistry and Crystallography. Candidates with a promising track record in the relevant areas and a proven publication record in international journals are encouraged to apply. Appointment will be made at a level appropriate to the successful applicant's qualifications, experience and in accordance with classification standards for each level. *Contact:*Margaret billsmargaret.bi...@monash.edu mailto:margaret.bi...@monash.edu
[ccp4bb] Protein-DNA modelling
Can anyone recommend a good modelling program to use to model protein-DNA interactions. I have tried Haddock. Any suggestions? Best Careina
