[ccp4bb] protein polymerization
Dear All, My proein is polymerzated and elutate in the void volum when runnning gel filtration Superdex 200. I can get a small crystal after a lot of optimization. But the resolution is still very low (about 8A ). I try to find the sites involved in polymerization. Is there some software to predicat the amino acids which are involved in protein polymerization? And All suggestion to the crystal optimization are welcome. Thank you. Best Regards, Lisa
[ccp4bb] AW: [ccp4bb] protein polymerization
Dear Lisa, the first thing to check is whether the polymerization is due to disulfide bond formation. If you run a gel of your protein with one sample boiled in the presence of b-mercapto ethanol and one sample boiled in the absence of bME, this should tell you whether disulfide links are the culprit. If disulfide links are the problem you could try adding DTT or TCEP to all your buffers, or to mutate the suspicious cysteines away. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von LISA Gesendet: Montag, 17. Februar 2014 10:58 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] protein polymerization Dear All, My proein is polymerzated and elutate in the void volum when runnning gel filtration Superdex 200. I can get a small crystal after a lot of optimization. But the resolution is still very low (about 8A ). I try to find the sites involved in polymerization. Is there some software to predicat the amino acids which are involved in protein polymerization? And All suggestion to the crystal optimization are welcome. Thank you. Best Regards, Lisa
[ccp4bb] new release of Mosflm iMosflm version 7.1.0
Hi folks We are pleased to announce a new release of Mosflm iMosflm; there are quite a few changes (a non-exhaustive list is at the end of this message). The default web-pages for both programs will now lead you directly to the new versions; http://www.mrc-lmb.cam.ac.uk/harry/imosflm and http://www.mrc-lmb.cam.ac.uk/harry/mosflm *-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*- *-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*- Changes since 1.0.7 / 7.0.9 iMosflm has been re-numbered to 7.1.0 so that its index coincides with that for Mosflm. There was no version of iMosflm between 1.0.7 and 7.1.0. 1. It is now possible to attempt to index and integrate images showing multiple lattices. This is an initial implementation and will only work in straightforward cases, for example the difference in orientation of the lattices must be greater than 1-2 degrees. Also at present it is only possible to use those reflections that are not overlapped when using the data in any downstream processing, and this will usually mean a significant drop in completeness. More details are given below. 2. It is now possible to distribute the integration task over multiple cores (parallel processing) while retaining the graphical output (only for OSX and Linux, not for Windows). 3.It is now possible to merge multiple MTZ files in QuickSymm/ QuickScale. In addition, it is possible to have some control over the scaling (exclude batches, reset resolution limits etc). 4. When completed, the results from the beam-search in the Indexing step are now sorted on the positional residual (discriminating against solutions with larger unit cells) to make it easier to identify the correct solution. 5. A traffic light system of warnings has been introduced, to make it easier to identify when there is a serious problem with processing. 6. Improved spot finding for very small spot sizes (eg in situ crystals with the Pilatus detector). 7. Small changes to autoindexing. 8. Resolution ranges to exclude from processing can now be defined explicitly, to avoid the severe loss in completeness that results from excluding all possible resolution shells for ice diffraction. In addition, anisotropic resolution limits may be specified. 9. It is now possible to define the detector omega angle for non- standard installations. 10. A site file can be used to specify experimental parameters that are known to be incorrect in the image header. This saves having to correct these parameters in every session of iMosflm. The site file has format . Allowed keywords: WAVELENGTH DISPERSION POLARISATION [PINHOLE | MIRRORS | MONO | SYNCHROTRON ] DIVERGENCE BEAM DISTANCE DISTORTION TILT DISTORTION TWIST GAIN DETECTOR REVERSEPHI DETECTOR OMEGA 11. A site file can be read or saved from the Session menu, or on startup using: imosflm --site A message will given in the launch window if any error in the keyword or value is detected. Other command line options are also available, listed by typing imosflm --help. 12. The CCP4 Uniqueify script is run automatically after TRUNCATE, providing an MTZ file with Rfree flags included. 13. QtRView replaces BAUBLES to present results from POINTLESS/ AIMLESS/TRUNCATE, so this no longer depends on a Web browser. 14. Multiple small bug fixes. Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
Re: [ccp4bb] AW: [ccp4bb] protein polymerization
*** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Dear Lisa, Good think is that your protein is crystallizing and diffracting X-rays. I am not sure if your protein is polymerizing. This could be a simple precipitation. Please check your pH, salt conditions or if you require some co-factors etc in addition to checking with bME as suggested by Herman. Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: herman.schreu...@sanofi.com To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 17 Feb 2014 10:06:18 + Subject: [ccp4bb] AW: [ccp4bb] protein polymerization *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Dear Lisa, the first thing to check is whether the polymerization is due to disulfide bond formation. If you run a gel of your protein with one sample boiled in the presence of b-mercapto ethanol and one sample boiled in the absence of bME, this should tell you whether disulfide links are the culprit. If disulfide links are the problem you could try adding DTT or TCEP to all your buffers, or to mutate the suspicious cysteines away. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von LISA Gesendet: Montag, 17. Februar 2014 10:58 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] protein polymerization Dear All, My proein is polymerzated and elutate in the void volum when runnning gel filtration Superdex 200. I can get a small crystal after a lot of optimization. But the resolution is still very low (about 8A ). I try to find the sites involved in polymerization. Is there some software to predicat the amino acids which are involved in protein polymerization? And All suggestion to the crystal optimization are welcome. Thank you. Best Regards, Lisa --- End of Original Message --- - Note: The information contained in the e-Mail message and/or attachments to it may contain confidential or privileged information. If you are not the intended recipient, any dissemination, use, review, distribute, prinitng or copying of the information contianed in this e-Mail message and/or attachments to it are strictly prohibited. If you have received this communication in error. Please notify us by reply e-Mail or telephone and immediately and permanently delete the message and any attachment. Thank you.
[ccp4bb] Twilight update
Dear Friends of the Twilight, the annual update of the twilight data base is now available: Download: http://www.ruppweb.org/twilight/default.htm Readme: http://www.ruppweb.org/twilight/Readme.htm Papers: http://journals.iucr.org/d/issues/2013/02/00/issconts.html http://journals.iucr.org/f/issues/2013/02/00/issconts.html Happy browsing in the twilight zone. EP, CXW, and BR PS: we appreciate reports of false positives for program improvement. Bernhard Rupp k.-k. Hofkristallamt Ordo Militum Vindicis Crystallographiae Vista, CA 92084 001 (925) 209-7429 mailto:b...@ruppweb.org b...@ruppweb.org mailto:b...@hofkristallamt.org b...@hofkristallamt.org http://www.ruppweb.org/ http://www.ruppweb.org/ ---
[ccp4bb] Gordon Conference on Diffraction Methods in Structural Biology
Dear all, The biannual Gordon Research Conference in Structural Biology, accompanied by the first Gordon Research Seminar, will take place in the last week of July at Bates College, New England, a few hours drive from the IUCr meeting that follows in the first week of August. The theme for the GRC is Faster, Smaller, Better: Novel Technologies for Diffraction Experiments in Molecular Biology and Drug Discovery and of the GRS (which precedes the GRC and is targeting young scientists giving them an opportunity to present their own work to their peers prior to the meeting) Towards Integrative Structural Biology. We have a truly fantastic line of speakers and discussion leaders, including John Kuriyan , Randy Read, Ana Gonzales, Ilme Schlichting , Henry Chapman , John Spence, Graeme Winter , Aina Cohen, Thomas Schneider , Gwyndaf Evans , Bob Fischetti , Flora Meilleur, Janet Newman , John Hunt , Michael Duszenko , Jose Antonio Marquez, Paul Adams , Clemens Vonrhein , Airlie McCoy , Brent Nannenga, Zbyszek Dauter , Tom Terwilliger , Garib Murshudov , Gerard Kleywegt , Paul Emsley, Dmitri Svergun , Peter Zwart , Michael Hammel , Lois Pollack, Elspeth Garman, Lisa Keefe , Gary Gililland , Aydnan Achour and Giovanna Scapin. For more details visit our web site: https://www.grc.org/programs.aspx?year=2014program=diffrac And here is the largely unavoidable motivational speech for anyone interested to my highly biased personal view: I first went to this meeting in 1998, as a young post-doc, presenting results that later led to the 'ARP/wARP' software: in fact, I had changed my slides (for the younger audience: slides were pieces of photographic film that were typically projected as mirror images of what you really wanted) a month or so before the meeting, as we got our very first models auto-built. I thought it was the most educational and exciting meeting I have ever been to, and in many ways it has shaped my research plan and my career (and my hate for golf). I wish I could also say that I never missed any of the subsequent meetings, but courtesy of the US Visa authorities and the Greek Army, I actually did a miss a couple. However, I still find them as exciting as so many years ago, but for a whole different set of scientific reasons: the diffraction methods landscape is changing rapidly, new machines and concepts make possible experiments that we do not even know what they are going to be! I am honored to be chairing this year's edition, and I hope that I will hear from at least one of you, what I had heard from a few before: this is the best meeting I have been in my life. Science aside, I am glad there is no golf course nearby the Bates College site where we are holding this meeting for the last decade, I am equally happy for the Great Outdoors site where we do the mid-week excursion, and I am looking forwards to the football (sorry: soccer) and basketball games. The Bates College boasts an excellent auditorium for the talks, a truly outstanding lounge for the poster sessions (that are always accompanied by drinks, an observation that might partially explain the tendency to finish well after midnight in a very positive spirit) and a somewhat confusingly and an unexpectedly good quality restaurant in the very friendly College site. A limited amount of (partial) bursaries to young scientists will be available for this year - when this becomes definitive we will post the news. I should also mention that one session will host eight seminars that will be selected from the poster sessions, giving all particpants the opportunity to present their own research! In the meantime, I am looking forward to welcome you all at the meeting and I hope you will register ... today! Best regards, Tassos Anastassis (Tassos, Perrakis, Principal Investigator , Staff Member Department of Biochemistry (B8, Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile , SMS: +31 6 28 597791
[ccp4bb] Ask for recommendation: crystal screening kit
Dear CCP4 friends, Sorry first for the out of topic request. We are going to buy more crystal screening kit, however, we are only familiar with some popular kits which we already have in the lab (listing below). Do you have your favourite kits which can share with us? Our lab more focus on enzymes and DNA/RNA binding proteins. Thank you. The kits we have in the lab: 1. Crystal Screen 1 and 2 (Hampton Research) 2. Index (Hampton Research) 3. Natrix 1 and 2 (Hampton Research) 4. PegRx 1 and 2 (Hampton Research) 5. Peg-Ion 1 and 2 (Hampton Research) 6. JCSG+ (Molecular Dimensions) 7. Morpheus (Molecular Dimensions) 8. Midas (Molecular Dimensions) Kind regards, Wenhe
Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein
Hi Raji- To echo what John Lee said, we found that we had to maintain a ratio between 1:2 and 1:10 Ulp1:substrate for several different target proteins, none of which were membrane proteins however. So, I would definitely try maybe a 1:5 ratio and see what happens. This wasn't too cost prohibitive as we were expressing a His-tagged Ulp1 and could afford to use quite a bit when we needed to. We also found that having 5-10% glycerol and 0.2% NP-40 (Igepal) enhances cleavage and helped keep our newly cleaved target protein from precipitating. Most importantly, we discovered that we could not simply scale up the volume of the cleavage reaction after running small volume pilot experiments. So, we would set up numerous 10 mL cleavage reactions in 15 mL conical tubes, incubate, and every 30 minutes or so we would invert the tubes to mix (we were mainly incubating at 30 C for ~3 hours). Doing it this way we found we went from ~50% cleavage to ~80-90%. I realize that duplicating that is impractical in your case but perhaps you could set up an orbital or shaker at 4C and gently keep the solution moving for the incubation. By the way, we tested dialysis and on-column cleavage and neither was better than the above method in our hands. Happy to discuss further or answer any questions off board. Best- Brad On Mon, Feb 17, 2014 at 10:50 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Thanks very much for your helpful suggestions. Several folks have suggested switching to TCEP. I am also going to increase the molar ratio of Ulp1 in the cleavage reaction like John Lee has suggested since I did not think of the effect of DDM on Ulp1. (Everything seemed to work in the case of the control SUMO-tagged soluble protein in presence of DDM so I didn't think of that as an issue at the time.) If these tweaks do not end up working in my case, I'm inclined to make and test a construct with a longer linker region, like several people have suggested. Many thanks again! Raji On Sun, Feb 16, 2014 at 10:58 AM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Workshop on Strategic pipeline planning: from sample preparation to 3D structure determination with bio SAXS and other biophysical techniques
Dear colleagues, with this message I would like to announce our workshop entitled: *Strategic pipeline planning: from sample preparation to 3D structure determination with bio SAXS and other biophysical techniques* to be held at the National Hellenic Research Foundation (NHRF) in Athens from April 5th to April 10th, 2014. *The workshop *will provide training on creating a roadmap towards determining the 3D structure of a macromolecular target including troubleshooting and rational decision-making with emphasis on Bio SAXS. The sessions will include: - *Structural Biology in Europe 2014 onwards *(ESFRI actions Instruct - a success story, Large scale facilities in Europe Opportunities for transnational access) - *Perspectives in structural biology *(An overview of additional main stream methods for structural biology: X-ray crystallography, NMR, X-ray imaging, ΕΜ) - *Sample preparation *(Key objectives and how to benchmark the success of a strategy, bottlenecks and troubleshooting, decision making on method selection for 3D structure determination) - *Bio SAXS at the forefront of structural biology *(Basic principles of SAXS, Supporting biophysical techniques e.g. Dynamic Light Scattering, ITC, SAXS data analysis software, in vitro and in silico SAXS challenges) - *Career development* (Mobstacles and prospects for young scientists in the frame of Horizon 2020) *Target Group: *PhD students and post-doctoral scientists, currently working in the structural biology field and have already some experience in working with macromolecules. Experienced researchers will also be considered but priority will be given to younger scientists. A total of 25 participants will be accepted in the course. *Selection process*: The Biostruct-X and local organizers will be responsible for the selection process to ensure that equivalent criteria will be applied. The applicants will be expected to send - a CV (max. 2 pages), - a motivation letter (max. 1 page) explaining why they wish to participate and what they expect from the course and - a reference letter. * Deadline: Wednesday, March 5th, 2014 ** *Benefits: *Participants that have been accepted to the course are offered : - to *send their protein samples (90% pure) to the Sample Preparation and Characterization (SPC) facility at the EMBL in Hamburg*. The SPC will offer a preliminary sparse matrix screen that can be customized to the particular sample, complementary characterization by thermal shift assay, dynamic light scattering and mass spectrometry. *The preliminary results of this characterization will be discussed during the workshop*. For more information, please visit the SPC website: https://htx.embl-hamburg.de/htxlab/ - *a limited number of fellowships* to be awarded to participants coming from INSTRUCT member countries (Belgium, Czech Republic, France, Germany, Israel, Italy, Netherlands, Portugal, Spain, Sweden, United Kingdom) awarded by INSTRUCT (http://www.structuralbiology.eu). *Host:* The event will be hosted by NHRF, coordinator of Instruct-EL, the national distributed research infrastructure that functions as a hub for structural biology in South East European area and Cyprus. *The workshop is organized in conjunction with the International Conference on Research Infrastructures (ICRI-2014) that will also be held in Athens 2-4 April, http://www.icri2014.eu http://www.icri2014.eu * *Registration fee*: EURO 50 for academic participants and EURO 1000 for participants coming from industry. *Confirmed speakers in alphabetical order include:* P. Agianian, T. Bergfors, J-M. Carazo, M. Graewert, M. Kokkinidis, P. Konarev, R. Meijers, G. Nounesis, E. Pereiro, M. Petoukhov, S. Pispas, E. Saridakis, S. Savvides, Th. Schneider, G. Spyroulias, P. Shaw-Stewart, Ef. Stratikos, D. Stuart, D. Svergun, C.E. Vorgias, M. Weiss, M. Wilmanns *Organizing committee:* *Local organizers :**National Hellenic Research Foundation (NHRF, host) *(E.D. Chrysina, V. Papadimitriou, G. Sotiroudis, A. Xenakis, M. Zervou, P. Zoumpoulakis) *Hellenic Crystallographic Association *(I.M. Mavridis, S.E. Zographos) *ICRI-2014 organizers*: *European Commission*, Directorate General for Research Innovation (Octavi Quintana Trias, Director of ERA policy) *Athena- Research and Innovation Center (on behalf of the Greek presidency/GSRT) *(Yannis Ioannidis, President and General Director) *Lead contact:* Dr Evangelia Chrysina, echrys...@eie.gr *More information will appear shortly on NHRF website*: http://www.eie.gr/index-en.html The workshop is supported by: - *European Commission, Seventh Framework Programme (FP7)* - *Biostruct-X *(http://www.biostruct-x.eu) - *INSTRUCT *(http://www.structuralbiology.eu) - *DECTRIS *(http://www.dectris.com) - *Douglas Instruments *(http://www.douglas.co.uk) - *Rigaku *(http://www.rigaku.com) - *Anelis Analytical
