Re: [ccp4bb] refinement protein structure
Dear Tom in addition to the very valuable input you have received already, I would like to point out that based on the fact that the I/sigma_I of your data is above 3 in the highest resolution shell, you will likely be able to push the resolution limits of your analysis to go beyond 1.4 angs resolution. I would definitely enoucrage you to do so even if Rmeas values end up being higher than what common practice and rules of thumb may prescribe. Recent work by Karplus and Diederichs (Science 336, 1030 (2012)) and an accompanying editorial by Phil Evans (Science 336, 986 (2012)) will provide the necessary food for thought to proceed. Very best wishes Savvas Savvas Savvides Unit for Structural Biology, L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html On 27 Mar 2013, at 17:22, Tom Van den Bergh tom.vandenbe...@student.kuleuven.be wrote: Dear members of ccp4bb, I need some help with the refinement of my structure of a variant of mRFP (monomer red fluorescent protein, sequence in attachment). I have done molecular replacement with phaser with model 2VAD of protein database. Then i have done some model building phenix.autobuild. (2 pdb's (overall...), freeR flags and log file attached) When i refine with phenix.refine my structure i get a R-value of 0,42 which is still way too high. (redfluorescent protein.pdb, .mtz and logfile attached) When i look at the structure in coot i find many unmodelled blobs and many outliers in density analysis and rotamer analysis. The problem is that there are so many problems with my structure, that i dont know where to begin. Could you try some refinement for me, because this is first structure that i need to solve as a student and i dont have too many experience with it. Greetings, Tom overall_best.pdbexptl_fobs_phases_freeR_flags.mtzoverall_best_placed.pdbredfluorescentprotein_refine_10.pdbredfluorescentprotein_refine_10.mtzmRFPsequentiezondertag.fastaredfluorescentprotein_refine_10.logAutoBuild_run_6_1.log
Re: [ccp4bb] delete subject
Hello, I stayed away from this thread until now - the major reason being that I was fitting snugly under my quilt. However I feel compelled to react now: placing your data in a public repository (thereby proving that you did the work) also means that a colleague, friend or whatever can and will publish your work for you. Once your work has been published you cannot publish it again, you did the work and the colleague, friend or whatever has in fact appropriated your work. In the world of dreams I was living in until a few moments ago (it was night time), this is perhaps the way we should act. In the real world we live in, even your colleague upstairs will publish your work if he / she has a chance to do it because by doing so he / she will improve his / her career while ensuring that yours doesn't take off. Fred. On 28/03/13 01:34, mjvdwo...@netscape.net wrote: Earlier today, I thought this and did not write it. It is a slightly different theme on your suggestion: I hear there are now (but have not seen examplesof) journals (web sites) where you do exactly what Tom did: you put your data there, which proves that you did the work (first) and you do not worry about the fact that you are making it public before formal publication, because making data public is the reason why you got the data in the first place. And nobody can claim to have done the work, because everybody knows that someone else was first - the web site is proof. Theresults are not peer-reviewed of course (even though, in the case of CCP4, things are inherently peer-reviewed to some extent, that is what he asked us to do). And I hear that there are now journals that will accept references to such web sites. Freely sharing unpublished data on a public forum might well be the future, even if in our corner of science this is not yet commonplace. The pivotal point to Tom is that he can learn from the suggestions that have been made. I hope he will. I actually hope that he will follow up on the suggestions (privately maybe). Unlike some, I do not feel that it was bad to find a big file in my inbox, this is what move to is for. I think my reaction was ouch, he did not want to do what he just did and it cannot be undone. But maybethis is not true. There is definitely value in sharing preliminary data, especially for junior people. To have such a function as part of CCP4 might be a very good suggestion, but I agree with you that perhaps it should not land in its full glory in everyone's mailbox. Mark -- Fred. Vellieux (B.Sc., Ph.D., hdr) ouvrier de la recherche IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] delete subject
Dear all, Let me start by apologizing for finally making this email longer than I intended - I did not have the time to make it shorter. I must say I am humbled by the amount of positive energy and constructive thinking that Bill has. That must explain also how he manages to keep up a fantastic resource for the community, what is a largely thankless task with little academic reward, but still so helpful. His response did get me thinking, as his opinions often do. In principle, in an era of information sharing, why don't we indeed solve structures with the collective brain of world crystallographers? We could share data, and educate people, and also get better structures at the end of the day. As indeed many people are in a small institution somewhat off the beaten path - I work in a cancer research institute after all and I am the weirdo here - and as indeed I see my knowledge getting obsoleted in a steady pace, this idea sounds great! However, its a bit like 'true' socialism - a grant idea, that we may find it will not work too well in practice, at least not under the constraints of human nature. I will keep advocating the greatness of ccp4bb. Its a fantastic resource, which is made truly amazing by the many questions that are being asked, and keep educating everybody. But, at the same time, I will keep advocating the usefulness of the typical constructions we have in science, whereupon people work in teams. These teams are there to share experience and help each other with overlapping expertise. Why such a basic question (and others in the past) need to come out of the 'team'? Is there no competence within the team to address it, or is there no correct communication? In either case, should we as scientists encourage such teams with low-level competence? May I remind you that Tom is in Lueven/Belgium, a large and outstanding University, with at least two very competent (and friendly) crystallographers in campus. Does the fact he has to post the ccp4bb with a basic question testify for a complete failure of his supervisor to either help him or get other people on site to help him? Should such supervisors be left to guide students? I have nothing against sharing data. I am the fool that submits data at the same time as I submit my paper, a practice that is followed by surprisingly a few people, as most people wait a few weeks until the paper is accepted to submit their structure (some data mining shows that 1/3 of the PDB entries associated with papers even in journals like Acta D are only deposited to the PDB at least a week after the paper submission date!.. no think what this % is in other journals). And the mild consequence of this is that somebody picks the structure up, panics to be scooped, submits his/her story, and scoops you while your paper is being rejected for reasons that are not connected to the structure (I am not implying foul play here, but suggesting a consequence of basic openness). Are we finally, at the end, with this open and sharing spirit, encouraging people to think that crystallography is too trivial? It has once been said in this bb, that 'solving a structure is trivial in the same way that climbing mountain Everest is trivial: it has been down before, its being done now, and it will be done again, by many well-trained and determined people'. Many people have read and trained for this task. If you do not read a couple of books and train before attempting the climb, and you send an email asking the everestbb 'does anybody know how to open this oxygen valve?' you are asking for trouble though … and the people that let you attempt the climb without that knowledge, are also in the wrong. The end result of this open and sharing spirit, which downgrades the importance of competence in major methodologies like X-ray crystallography, was summarized recently is some text I recently got by email from Brussels: ... advanced methods for X-ray crystallography and Electron Microscopy is a very narrow field that will limit the employability of the graduates There are the wise words of the referees of a joined grant (with 10 other people from Europe) advocating to educate students to get an in-depth PhD-level training in crystallography and in EM. Maybe that explains my grumpiness. Or, as the crystallographer previously known as DVD mailed me in private welcome to the club of grumpy old men. Maybe I am just being grumpy. Or I am justifiably worried. A. PS For the record I admire Tom's spirit and courage - he is the kind of guy I would hire for a PhD (not that he will ever want to work with me any more). I am less impressed by the team and his supervisors, as it stands, and without knowing all the details of what might be behind this. On Mar 28, 2013, at 1:04, William G. Scott wrote: Dear Tom et al: Although arriving too late to participate in the snark-fest, it occurred to me that maybe this is almost exactly how we
Re: [ccp4bb] delete subject
I fully agree that questions should keep coming. In addition it might be worth pointing out that CCP4 (and others) organise great crystallographic schools which are ideal for this sort of problem. Most of the schools ask you to bring your own data (!) and you can sit down with developers and experts who will help at length with practical and theoretical questions. Roberto From my iPhone On 28 Mar 2013, at 04:47, Frank von Delft frank.vonde...@sgc.ox.ac.ukmailto:frank.vonde...@sgc.ox.ac.uk wrote: mistake? I beg to differ, violently: a student had an honest question and did exactly what the ccp4bb exists for: posted his question there. Moreover, when asking he showed he had thought about it, and provided complete background -- that's what we all want, right? * I disagree with Tassos: the email was not rude, on not one of the counts he listed. (I concede he may have had a bad day... I had one on Monday :-) * I disagree (slightly) with Tim: the teasing was not malicious. * And I share Mark's dream... Students: please don't stop asking your questions here!!! phx. On 27/03/2013 22:47, VAN RAAIJ , MARK JOHAN wrote: Dear Tom, don't feel too bad about it - everyone can make a mistake. Some of the replies give crystallographic tips that may be useful to other beginning and not-so-beginning crystallographers. Although I agree the attachments to the first mail would perhaps better be deleted from the records. Greetings, Mark Quoting Tom Van den Bergh mailto:tom.vandenbe...@student.kuleuven.betom.vandenbe...@student.kuleuven.bemailto:tom.vandenbe...@student.kuleuven.be: Is it possible to delete my post: refinement protein structure from ccp4 bb, i get too many bad reactions. I think its bettter to just delete the whole topic. Greetings, Tom
Re: [ccp4bb] delete subject
All, I personally would feel awkward at depositing my hard fought data before the associated paper is accepted. However, a recent paper from our institution (I was not part of that study) originated from precisely the model that has been suggested in this thread: http://www.nejm.org/doi/full/10.1056/NEJMoa1107643 Given the success of various 'open source' endeavours in recent years, I'm slowly coming around to a view that with smart procedures in place, an 'open data' approach could indeed help to solve structures that would otherwise remain unsolved, unpublished and unused. Klaus On 28 Mar 2013, at 08:46, Anastassis Perrakis wrote: Dear all, Let me start by apologizing for finally making this email longer than I intended - I did not have the time to make it shorter. I must say I am humbled by the amount of positive energy and constructive thinking that Bill has. That must explain also how he manages to keep up a fantastic resource for the community, what is a largely thankless task with little academic reward, but still so helpful. His response did get me thinking, as his opinions often do. In principle, in an era of information sharing, why don't we indeed solve structures with the collective brain of world crystallographers? We could share data, and educate people, and also get better structures at the end of the day. As indeed many people are in a small institution somewhat off the beaten path - I work in a cancer research institute after all and I am the weirdo here - and as indeed I see my knowledge getting obsoleted in a steady pace, this idea sounds great! However, its a bit like 'true' socialism - a grant idea, that we may find it will not work too well in practice, at least not under the constraints of human nature. I will keep advocating the greatness of ccp4bb. Its a fantastic resource, which is made truly amazing by the many questions that are being asked, and keep educating everybody. But, at the same time, I will keep advocating the usefulness of the typical constructions we have in science, whereupon people work in teams. These teams are there to share experience and help each other with overlapping expertise. Why such a basic question (and others in the past) need to come out of the 'team'? Is there no competence within the team to address it, or is there no correct communication? In either case, should we as scientists encourage such teams with low-level competence? May I remind you that Tom is in Lueven/Belgium, a large and outstanding University, with at least two very competent (and friendly) crystallographers in campus. Does the fact he has to post the ccp4bb with a basic question testify for a complete failure of his supervisor to either help him or get other people on site to help him? Should such supervisors be left to guide students? I have nothing against sharing data. I am the fool that submits data at the same time as I submit my paper, a practice that is followed by surprisingly a few people, as most people wait a few weeks until the paper is accepted to submit their structure (some data mining shows that 1/3 of the PDB entries associated with papers even in journals like Acta D are only deposited to the PDB at least a week after the paper submission date!.. no think what this % is in other journals). And the mild consequence of this is that somebody picks the structure up, panics to be scooped, submits his/her story, and scoops you while your paper is being rejected for reasons that are not connected to the structure (I am not implying foul play here, but suggesting a consequence of basic openness). Are we finally, at the end, with this open and sharing spirit, encouraging people to think that crystallography is too trivial? It has once been said in this bb, that 'solving a structure is trivial in the same way that climbing mountain Everest is trivial: it has been down before, its being done now, and it will be done again, by many well-trained and determined people'. Many people have read and trained for this task. If you do not read a couple of books and train before attempting the climb, and you send an email asking the everestbb 'does anybody know how to open this oxygen valve?' you are asking for trouble though … and the people that let you attempt the climb without that knowledge, are also in the wrong. The end result of this open and sharing spirit, which downgrades the importance of competence in major methodologies like X-ray crystallography, was summarized recently is some text I recently got by email from Brussels: ... advanced methods for X-ray crystallography and Electron Microscopy is a very narrow field that will limit the employability of the graduates There are the wise words of the referees of a joined grant (with 10 other people from Europe) advocating to educate students to get an in-depth PhD-level
[ccp4bb] process data from two crystals in-house data
Dear all I have two data sets, 75 frames each from crystals of the same protein - same cell parameters/space group (P4). I could process them seperately each yielding 90% completeness but with poor multiplicity ( 2 ) If I merge the data sets using CAD, I loose the data reduction statistics of the combined data set. I do not have HKL2000. Which (free) tool can handle two different diffraction data sets, process them together to give a single final data statistics. Thanks and regards Thiyaga S. Thiyagarajan Centre of Excellence in Bioinformatics School of Biotechnology Madurai Kamaraj University Madurai - 625021 Ph: +91-9159224881 (cell)
Re: [ccp4bb] process data from two crystals in-house data
Hello, I would recommend to you XDS package, particularly XSCALE program (link to wiki page): http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Xscale 28.03.2013 14:08, S. Thiyagarajan ?: Dear all I have two data sets, 75 frames each from crystals of the same protein - same cell parameters/space group (P4). I could process them seperately each yielding 90% completeness but with poor multiplicity ( 2 ) If I merge the data sets using CAD, I loose the data reduction statistics of the combined data set. I do not have HKL2000. Which (free) tool can handle two different diffraction data sets, process them together to give a single final data statistics. Thanks and regards Thiyaga S. Thiyagarajan Centre of Excellence in Bioinformatics School of Biotechnology Madurai Kamaraj University Madurai - 625021 Ph: +91-9159224881 (cell) -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] process data from two crystals in-house data
Sorry I am mentioning non-ccp4 software here: XDS processing (the 2 data sets separately); AIMLESS / TRUNCATE and checking the truncate plots to see if there are frames (Batches) that should better be discarded; if need be, XDS processing again, XSCALE, XDSCONV (option CCP4_F if you only want F, SIGF and FreeRFlag). HTH, Fred. On 28/03/13 11:08, S. Thiyagarajan wrote: Dear all I have two data sets, 75 frames each from crystals of the same protein - same cell parameters/space group (P4). I could process them seperately each yielding 90% completeness but with poor multiplicity ( 2 ) If I merge the data sets using CAD, I loose the data reduction statistics of the combined data set. I do not have HKL2000. Which (free) tool can handle two different diffraction data sets, process them together to give a single final data statistics. Thanks and regards Thiyaga S. Thiyagarajan Centre of Excellence in Bioinformatics School of Biotechnology Madurai Kamaraj University Madurai - 625021 Ph: +91-9159224881 (cell) -- Fred. Vellieux (B.Sc., Ph.D., hdr) ouvrier de la recherche IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] process data from two crystals in-house data
Hi - You can use Xia2 or, you can use Pointless (Find or Match Laue group) to combine the two datasets after Mosflm in one MTZ with common correct origin etc, and then use SCALA to scale them together. For more info: http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Data_processing_with_CCP4 Its btw the same procedures (albeit for a different problem) as suggested a week ago under the subject Scaling with SCALA high and low resolution data sets A. On Mar 28, 2013, at 11:08, S. Thiyagarajan wrote: Dear all I have two data sets, 75 frames each from crystals of the same protein - same cell parameters/space group (P4). I could process them seperately each yielding 90% completeness but with poor multiplicity ( 2 ) If I merge the data sets using CAD, I loose the data reduction statistics of the combined data set. I do not have HKL2000. Which (free) tool can handle two different diffraction data sets, process them together to give a single final data statistics. Thanks and regards Thiyaga S. Thiyagarajan Centre of Excellence in Bioinformatics School of Biotechnology Madurai Kamaraj University Madurai - 625021 Ph: +91-9159224881 (cell) 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
Re: [ccp4bb] process data from two crystals in-house data
Hi I'd just integrate them individually with Mosflm, then merge the two MTZ files into one with Pointless(*), then scale with Aimless. (*) pointless hklin first.mtz second.mtz hklout pointless.mtz aimless hklin pointless.mtz hklout aimless.mtz should do the trick - pointless even works with a wild card if you have many mtz files to merge (not sure how many many can be, but it's certainly a lot more than 10), e.g. pointless hklin part_*.mtz hklout pointless.mtz Dead easy with normal CCP4 programs, RTM for pointless for further details. You can even do this in ccp4i *really easily*! On 28 Mar 2013, at Thu28 Mar 10:08, S. Thiyagarajan wrote: Dear all I have two data sets, 75 frames each from crystals of the same protein - same cell parameters/space group (P4). I could process them seperately each yielding 90% completeness but with poor multiplicity ( 2 ) If I merge the data sets using CAD, I loose the data reduction statistics of the combined data set. I do not have HKL2000. Which (free) tool can handle two different diffraction data sets, process them together to give a single final data statistics. Thanks and regards Thiyaga S. Thiyagarajan Centre of Excellence in Bioinformatics School of Biotechnology Madurai Kamaraj University Madurai - 625021 Ph: +91-9159224881 (cell) 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] delete subject
Dear ccp4 members, Thanks for all the help. I am now rebuilding my structure now based on all your advice (which was my intention all along). I am glad to see there are still scientists who dont only care about making and publishing their own data, but are always ready to help less experienced students with their experiments. Its also my personal believe that this approach can be a great help in scientific research as some people have already said and i am happy to see that i am not the only one who has this opinion. I think this is a good time to end the discussion. Kind regards, Tom Van: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] namens Steiner, Roberto [roberto.stei...@kcl.ac.uk] Verzonden: donderdag 28 maart 2013 10:08 To: CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] delete subject I fully agree that questions should keep coming. In addition it might be worth pointing out that CCP4 (and others) organise great crystallographic schools which are ideal for this sort of problem. Most of the schools ask you to bring your own data (!) and you can sit down with developers and experts who will help at length with practical and theoretical questions. Roberto From my iPhone On 28 Mar 2013, at 04:47, Frank von Delft frank.vonde...@sgc.ox.ac.ukmailto:frank.vonde...@sgc.ox.ac.uk wrote: mistake? I beg to differ, violently: a student had an honest question and did exactly what the ccp4bb exists for: posted his question there. Moreover, when asking he showed he had thought about it, and provided complete background -- that's what we all want, right? * I disagree with Tassos: the email was not rude, on not one of the counts he listed. (I concede he may have had a bad day... I had one on Monday :-) * I disagree (slightly) with Tim: the teasing was not malicious. * And I share Mark's dream... Students: please don't stop asking your questions here!!! phx. On 27/03/2013 22:47, VAN RAAIJ , MARK JOHAN wrote: Dear Tom, don't feel too bad about it - everyone can make a mistake. Some of the replies give crystallographic tips that may be useful to other beginning and not-so-beginning crystallographers. Although I agree the attachments to the first mail would perhaps better be deleted from the records. Greetings, Mark Quoting Tom Van den Bergh mailto:tom.vandenbe...@student.kuleuven.betom.vandenbe...@student.kuleuven.bemailto:tom.vandenbe...@student.kuleuven.be: Is it possible to delete my post: refinement protein structure from ccp4 bb, i get too many bad reactions. I think its bettter to just delete the whole topic. Greetings, Tom
Re: [ccp4bb] delete subject
I am agree with Tassos. For me it is a quite stranger to think about hide the data to nobody see until the paper be published. In my opinion, a lot of data become obsolete (or even forgotten) because that one more experiment you need to publish a great paper (it some times takes years...). For me science has become an aggressive capitalist market where the paper is its currency, mainly in life sciences. But, due to the human nature, this sharing-data ideal world is far to become true and we need to try play this game to keep alive and try to change it for better. On the other hand, in my opinion, crystallography research are near of this ideal world in comparison with other areas (pharmacy, molecular and functional biology, ...) some proofs are the ccp4bb, workshops where you can get help to process your data, etc. So, please, do not stop to posting your questions and commentaries, they are very important to students like me. Cheers, *Andrey Fabricio Ziem Nascimento* PhD Student in Biochemistry/UNICAMP Biophysics and Structural Biology Group Brazilian Biosciences National Laboratory (LNBio) – CNPEM 2013/3/28 Klaus Fütterer k.futte...@bham.ac.uk All, I personally would feel awkward at depositing my hard fought data before the associated paper is accepted. However, a recent paper from our institution (I was not part of that study) originated from precisely the model that has been suggested in this thread: http://www.nejm.org/doi/full/10.1056/NEJMoa1107643 Given the success of various 'open source' endeavours in recent years, I'm slowly coming around to a view that with smart procedures in place, an 'open data' approach could indeed help to solve structures that would otherwise remain unsolved, unpublished and unused. Klaus On 28 Mar 2013, at 08:46, Anastassis Perrakis wrote: Dear all, Let me start by apologizing for finally making this email longer than I intended - I did not have the time to make it shorter. I must say I am humbled by the amount of positive energy and constructive thinking that Bill has. That must explain also how he manages to keep up a fantastic resource for the community, what is a largely thankless task with little academic reward, but still so helpful. His response did get me thinking, as his opinions often do. In principle, in an era of information sharing, why don't we indeed solve structures with the collective brain of world crystallographers? We could share data, and educate people, and also get better structures at the end of the day. As indeed many people are in a small institution somewhat off the beaten path - I work in a cancer research institute after all and I am the weirdo here - and as indeed I see my knowledge getting obsoleted in a steady pace, this idea sounds great! However, its a bit like 'true' socialism - a grant idea, that we may find it will not work too well in practice, at least not under the constraints of human nature. I will keep advocating the greatness of ccp4bb. Its a fantastic resource, which is made truly amazing by the many questions that are being asked, and keep educating everybody. But, at the same time, I will keep advocating the usefulness of the typical constructions we have in science, whereupon people work in teams. These teams are there to share experience and help each other with overlapping expertise. Why such a basic question (and others in the past) need to come out of the 'team'? Is there no competence within the team to address it, or is there no correct communication? In either case, should we as scientists encourage such teams with low-level competence? May I remind you that Tom is in Lueven/Belgium, a large and outstanding University, with at least two very competent (and friendly) crystallographers in campus. Does the fact he has to post the ccp4bb with a basic question testify for a complete failure of his supervisor to either help him or get other people on site to help him? Should such supervisors be left to guide students? I have nothing against sharing data. I am the fool that submits data at the same time as I submit my paper, a practice that is followed by surprisingly a few people, as most people wait a few weeks until the paper is accepted to submit their structure (some data mining shows that 1/3 of the PDB entries associated with papers even in journals like Acta D are only deposited to the PDB at least a week after the paper submission date!.. no think what this % is in other journals). And the mild consequence of this is that somebody picks the structure up, panics to be scooped, submits his/her story, and scoops you while your paper is being rejected for reasons that are not connected to the structure (I am not implying foul play here, but suggesting a consequence of basic openness). Are we finally, at the end, with this open and sharing spirit, encouraging people to think that crystallography is too trivial? It has once been said in
[ccp4bb] AW: [ccp4bb] cell disruptor / homogenizer - final summary
Since my previous summary on cell disruption equipment elicited a pretty clear response, I want to give a final update: Several methods and machines were mentioned (see below), but the Avestin Emulsiflex C5 is now the clear winner, with five happy users and no really negative comment. Thanks again to everyone for this helpful feedback! Happy Holidays Clemens Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Clemens Steegborn Gesendet: Dienstag, 26. März 2013 19:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] AW: [ccp4bb] cell disruptor / homogenizer - summary Dear colleagues, To summarize the feedback on cell disruptors: One person found the Emulsiflex performance worth the maintenance required, there were no comments at all on Fluidizer and TS 0.75! As alternatives, French press (Glenn Milles), beadbeaters (Biospec), Panda homegenizer (GEA Niro Soavi), and nitrogen cavitation in a pressure bomb (http://www.ncbi.nlm.nih.gov/pubmed/21041386) were suggested. Thanks for the comments and suggestions. Best Clemens Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Clemens Steegborn Gesendet: Montag, 25. März 2013 10:17 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] cell disruptor / homogenizer Dear colleagues, We are currently looking for new cell disruptor/homogenizer equipment, mainly for E.coli work. We currently have a Branson sonifier and a Microfluidics Fluidizer the latter one keeps causing trouble, and we think about a replacement. We previously also used an Avestin C-5 Emulsiflex, required quite some maintenance, and I inquired about a Constant Systems TS 0.75 and got mixed responses from users (not to mention their outrageous pricing). My question: What is your experience with Fluidizer, Emulsiflex, TS 0.75? Is there any other great (low maintenance, affordable especially concerning follow-up costs: repairs, replacement parts) equipment I missed? Thanks in advance for your comments Best regards Clemens
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Andrey, Due to our company policy, I cannot view the dropbox image, but the packing you describe is exactly what Edward Barry described and what I expected. Two molecules obey the 2-fold symmetry and one molecule is lying on a 2-fold with two possible superimposed orientations. The question is: are there frequent switches between both conformations such that X-rays diffracted from both conformations are interfering, or are there just larger chunks of crystal with either conformation A or B, behaving like separate crystals? In the first case, one has a crystal packing disorder and, as Eleanor was hinting at, some of the diffraction spots may look funny or smeared, in the second case one has twinning. I do not know the exact method sfcheck uses, but normally to detect twinning, programs do not look at symmetry but look at intensity distributions. E.g. in case of twinning, intensities are averaged and less reflections will have extreme (very high or very low) values and reflections will have more average values. I would expect that if you expand an untwinned P21212 data set to P21 and run the twin test, it still will come out untwinned. So if sfcheck claims the data is twinned, it most likely is and you have used the correct procedure. And in this case you should also process the data in the lower symmetry space group. What wonders me a little is that MR did not give clear solutions in P21 with twinned data. P21 is low symmetry and I would have expected 2 clear solutions, each corresponding to one of the two twin possibilities. Also, if the detwin procedure does what I think it does, it should not perform well if the twin fraction is very close to 0.5 (perfect twinning). As has been pointed out by others, you will have to generate the freeR flags in P21212 and expand them to P21, otherwise your free reflections are linked to working reflections and you will get artificially low free Rfactors. Also, Rfactors and free Rfactors will generally be lower if you use twin refinement. No reflections are discarded during twin refinement, what happens is that both twin orientations are taken into account during refinement (and for the calculation of Rfactors!). Congratulations, it looks like you have solved your problem! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrey Nascimento Sent: Thursday, March 28, 2013 12:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse - even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that solved the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present - P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.ukmailto:eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is
Re: [ccp4bb] delete subject
Ed, I very much agree with you. We've all had to learn that questions posted to ccp4bb and the ensuing discussions take on a life of their own. Once one posts a question on ccp4bb, there's no such thing as steering the direction of the discussion on the ccp4bb and there's no such thing as the equivalent of screaming Stop! Stop! Stop! on the ccp4bb. Also, I don't believe people simply woke up one day and posted irritating or mean comments to ccp4bb. Ed was spot on for why some folks reacted the way they did to the post so let's acknowledge that as well. I didn't get the impression that any of the replies suggested that students stop posting questions. There are many many students on this BB who are in small institutions without even the minimal help at arm's length and who get tons of help from posting questions to the ccp4bb. That situation is not all that distant in my own memory and I suspect for many other experts on this BB. But posting 10MB attachments and getting the entire ccp4bb community to crowdsource towards problem solving is all good, but only to a certain degree. It may be great to get things done quickly with the collective intellect of the ccp4bb but there comes a point when the correct answers may get fed back at such a rapid speed that if one doesn't go back and try to figure stuff out for oneself, including the reasons/theory/logic behind the answers/solutions that the community has posted, it may be to the detriment of one's own learning, especially if one is in the early stages of learning the subject matter. Cheers, Raji On Thu, Mar 28, 2013 at 9:09 AM, Ed Pozharski epozh...@umaryland.eduwrote: On Thu, 2013-03-28 at 12:15 +, Tom Van den Bergh wrote: I think this is a good time to end the discussion. As a general comment, discussions on boards like ccp4bb often digress and take direction different from you original intent. I may understand your desire to try to control the situation, but if people on this board feel that the questions of data sharing, student training, netiquette and proper choice of resolution cutoff are worthy of further discussion (that may not have much to do with specifics of your original request for assistance), it is their right too. What may have caused some extra grief is this unfortunate turn of phrase in your original post Could you try some refinement for me, because this is first structure that i need to solve as a student and i dont have too many experience with it. It goes a bit beyond the usual my R-values are too high what should I do question and may be instinctively construed as if you expect someone to actually do your work for you (I am sure that is not what you asked). So a bit of a vigorous reaction that you received likely results from misunderstanding your intent (albeit posting your data is very unusual and strengthens the impression) and perhaps misplaced feeling that you have abandoned attempts to resolve the problem independently too soon. I did *not* look at your data and therefore I may be completely wrong here, but it is my understanding that your actual issue was not realizing there could be more than one molecule in the asymmetric unit. More traditional route is to describe your situation in general terms and offer to provide data to those willing to take a closer look. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] delete subject
By coincidence this just landed in my Inbox: http://membercentral.aaas.org/multimedia/webinars/how-recruit-citizen-scientists-discovery So maybe after all Tom is way ahead of the rest of us in his structure-solving strategy - though I agree with others that his tactics need to be honed somewhat! Cheers -- Ian On 28 March 2013 14:43, Raji Edayathumangalam r...@brandeis.edu wrote: Ed, I very much agree with you. We've all had to learn that questions posted to ccp4bb and the ensuing discussions take on a life of their own. Once one posts a question on ccp4bb, there's no such thing as steering the direction of the discussion on the ccp4bb and there's no such thing as the equivalent of screaming Stop! Stop! Stop! on the ccp4bb. Also, I don't believe people simply woke up one day and posted irritating or mean comments to ccp4bb. Ed was spot on for why some folks reacted the way they did to the post so let's acknowledge that as well. I didn't get the impression that any of the replies suggested that students stop posting questions. There are many many students on this BB who are in small institutions without even the minimal help at arm's length and who get tons of help from posting questions to the ccp4bb. That situation is not all that distant in my own memory and I suspect for many other experts on this BB. But posting 10MB attachments and getting the entire ccp4bb community to crowdsource towards problem solving is all good, but only to a certain degree. It may be great to get things done quickly with the collective intellect of the ccp4bb but there comes a point when the correct answers may get fed back at such a rapid speed that if one doesn't go back and try to figure stuff out for oneself, including the reasons/theory/logic behind the answers/solutions that the community has posted, it may be to the detriment of one's own learning, especially if one is in the early stages of learning the subject matter. Cheers, Raji On Thu, Mar 28, 2013 at 9:09 AM, Ed Pozharski epozh...@umaryland.eduwrote: On Thu, 2013-03-28 at 12:15 +, Tom Van den Bergh wrote: I think this is a good time to end the discussion. As a general comment, discussions on boards like ccp4bb often digress and take direction different from you original intent. I may understand your desire to try to control the situation, but if people on this board feel that the questions of data sharing, student training, netiquette and proper choice of resolution cutoff are worthy of further discussion (that may not have much to do with specifics of your original request for assistance), it is their right too. What may have caused some extra grief is this unfortunate turn of phrase in your original post Could you try some refinement for me, because this is first structure that i need to solve as a student and i dont have too many experience with it. It goes a bit beyond the usual my R-values are too high what should I do question and may be instinctively construed as if you expect someone to actually do your work for you (I am sure that is not what you asked). So a bit of a vigorous reaction that you received likely results from misunderstanding your intent (albeit posting your data is very unusual and strengthens the impression) and perhaps misplaced feeling that you have abandoned attempts to resolve the problem independently too soon. I did *not* look at your data and therefore I may be completely wrong here, but it is my understanding that your actual issue was not realizing there could be more than one molecule in the asymmetric unit. More traditional route is to describe your situation in general terms and offer to provide data to those willing to take a closer look. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Repository of Unsolved Datasets in Macromolecular Crystallography?
Dear colleagues, I have followed (with great deal of interest) all the various expressed opinions about whether it may or may not be useful to have means whereupon challenging crystallographic data may be shared with the wider community of crystallographers in the hope that 'crowdsourcing' may eventually point to a solution that will yield biological insights. It seems that the general opinion is that such a means would be very useful. However, the crux of the matter is intellectual ownership of the resulting structures and the wider issues of co-authorship on subsequent publications. Someone has stated that it matters who 'solves' the structure first. Maybe I am gravely misguided about this, but I am under the impression that it does not. What matters is who publishes it first. Publications, and especially high-impact ones, remain pretty much the only criterion that is understood across all disciplines of scientific research. To the non-structural biologists sitting on funding panels it will not matter in the least if someone is acknowledged to have been the first to 'solve' a structure if they were not the first to publish it. In the case that has led to all of these discussions, it would seem to me that it would be an honourable thing for Tom's supervisors to ask Phil Jeffrey if he would like to be included as a co-author on this work when and if it is published. After all, his input and expertise would now enable Tom to get his structure to a publishable standard. Phil has provided valuable intellectual input into the research project. However, this presents a dilemma. Tom's supervisors have not, to our knowledge, directed Tom to seek such assistance through wide dissemination of data and their policies on what is the proper basis for co-authorship may be different to mine or indeed most of the research community. So what I would like to propose is that the CCP4 create a special repository on their website where people can upload their data if they wish to seek an expert opinion and get the structure 'solved'. To prevent potential embarrassment or dishonesty (i.e., stealing coordinates etc) a number of mechanisms can be put in place such as exist for the Innocentive Challenges (https://www.innocentive.com). For example, a brief outline of the data and the perceived issues with it can be given on the site for all to see, but if a crystallographer wishes to gain access to the data and have a crack at solving the structure or correcting whatever pathologies it may have and providing advice on structure solution they may have to digitally sign a legal form to identify themselves and to agree to the conditions stipulated by the person who deposited the data such as non-disclosure, statement on competing interests and whether they agree to waive their right to intellectual ownership and co-authorship. The full name of the principal investigator should be affiliated with the deposited data. Ideally, the person who deposits the data may exercise some choice in stipulating what conditions they would like to impose and may very well be only too pleased to make the expert a co-author if their contribution leads to successful structure determination. It seems such a repository would serve two purposes at the same time. On the one hand, it will put non-experts in direct contact with professional crystallographers to foster collaboration. It will also provide a rich vein from which to mine challenging datasets for methods developers who may have little interest in co-authorship on biology papers but can then use the data to improve software and computational tools that will benefit all of our community. With best regards, Eugene -- Dr Eugene Valkov MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 407840
Re: [ccp4bb] off topic, BN PAGE
Hi Careina, Gels certainly are convenient and powerful tools when they work. For normal native gel, do not forget that if you are using the Laemmlli buffer system (ie. tris-glycine), the actual pH during running is pH 9 and the ionic strength changes over time. There are other buffer systems, but you still need to be careful with the results. The other consideration is that when running a dimer in gel, if the dimer is not very stable, during a long run (for example, regular native gel often take hours), the dimer can dissociate significantly, resulting in a smear or total loss of the dimer band. I think if you can run gel-filtration experiments(a little more expensive in terms of equipment and protein consumption), you probably can draw conclusions with more confidence due to: 1) you can see the peaks in 20 minutes so even if the dimmer dissociates it will dissociate less in less time; 2) you can equilibrate the column in your buffers so there is no issue about the real condition you are testing. The drawback is that you can't test your different conditions in parallel. But with gels, your conditions only exist in the loading wells, when the proteins enter the gel and spend the 1-5 hrs there, your initial conditions are lost anyways. Zhijie From: Careina Edgooms Sent: Thursday, March 28, 2013 3:03 AM To: Zhijie Li Subject: Re: [ccp4bb] off topic, BN PAGE Hi Zhijie Thanks for the helpful reply. The attractive thing about BN PAGE (if it works of course) is that it is so quick, inexpensive and simple to perform. I am working with a protein that exists in the native state as a mixture of monomer and dimer. I wish to monitor the effects of various things on this monomer dimer equilibrium. The simplest way of doing this is to run a gel at the end of the experiment and quantify the bands with densitometry to check the effect on the monomer dimer equilibrium. When I run this protein on a BN PAGE gel it runs as 2 bands. I initially assumed these 2 bands to be a true representation of the monomer dimer equilibrium but now I am wondering if the coomassie could actually interfere with the contacts so that even if the protein was in a state where it was entirely dimeric, it would appear on the gel as 2 bands because the coomassie is interfering with the dimer interface. What I'm attempting to do now is run a regular native page without coomassie. Because my protein is very basic this means I will have to swap the electrodes. If this also shows 2 bands then I wonder if it is safe to assume that the BN PAGE works and coomassie does not interfere? Careina From: Zhijie Li zhijie...@utoronto.ca To: Careina Edgooms careinaedgo...@yahoo.com Cc: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, March 28, 2013 1:28 AM Subject: Re: [ccp4bb] off topic, BN PAGE Hi Careina, BN PAGE can be affected by many factors. Considering the complexity and the chance of winning and the amount of information you gain even when you win, I do not recommend fighting it. BN PAGE, like other gel-based methods, requires that your complex is fairly stable - not having a weak affinity and not a high dissociation rate. Also the complex formation should be compatible with the gel running condition: the pH and salts and other things. Coomassie itself may compete for some hydrophobic surfaces or positively charged residues on the proteins - in such case there's little you can do. If you see a band corresponding to the expected complex weight, then congratulations, BN gel might be your tool (with cautions though as you can also get false positives with BN gel). But if not, then my suggestion is to move to other techniques such as gel filtration, analytical ultracentrifugation, BiaCore, ITC and so on. I had a case in which I could confidently show complex formation with gel-filtration and Biacore, but not with BN gel. Zhijie From: Careina Edgooms Sent: Wednesday, March 27, 2013 5:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic, BN PAGE Hi Has anyone found the coomassie in a BN PAGE to be interfering with the oligomeric structure of their protein? If so, how did you deal with this? Thanks Careina
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Appu, I am sorry, I do not have the script file. I ran it basically from default parameters in CCP4 GUI. I just ran the sfcheck for 'check experimental data only' and I got the twin law and twin fraction from the output (post script file). So, I put these information on the detwin and ran it. I will send a .pdf file with the a print screen of ccp4 GUI. I am not a experienced crystallographer, but I hope it helps you. Good luck, Andrey 2013/3/28 Appu kumar appu.kum...@gmail.com Respected sir, I have same problem what you have. I am running the detwin on mtz file but it getting failed. Could you please tell me how you did this. If possible send me your script file. It will be a great help for me. Thank you in advance. Appu On 28 March 2013 05:10, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse – even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that “solved” the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present – P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the R-free set. But the map for third molecule (new molecule placed) are still very bad, even when a tried to reprocess data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 21 ASU) and its symmetry related on P21 shows a very good map, but the third molecule are almost completely wrong (~50 residues in 470 are placed in quite good map) and map does not have connectivity to build a new molecule (even in
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Herman, I would like to mention one more information, maybe I have forgotten. When a process the data in P21212 and run the sfcheck it do not appear to have twinning (even when a ran phenix Xtriage with older process data in P21212). I will send direct to your e-mail the .pdf file with sfcheck analysis to both space groups. Thank you very much. Andrey 2013/3/28 Andrey Nascimento andreynascime...@gmail.com Dear Appu, I am sorry, I do not have the script file. I ran it basically from default parameters in CCP4 GUI. I just ran the sfcheck for 'check experimental data only' and I got the twin law and twin fraction from the output (post script file). So, I put these information on the detwin and ran it. I will send a .pdf file with the a print screen of ccp4 GUI. I am not a experienced crystallographer, but I hope it helps you. Good luck, Andrey 2013/3/28 Appu kumar appu.kum...@gmail.com Respected sir, I have same problem what you have. I am running the detwin on mtz file but it getting failed. Could you please tell me how you did this. If possible send me your script file. It will be a great help for me. Thank you in advance. Appu On 28 March 2013 05:10, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, As I said in the latest topic, I could not model the third molecule. But when I superpose the two trimmers found in P1 MR solution (link below), I get the first two molecules perfect aligned and the third molecule inverted! (It is also possible to see the 2-fold axis and the third molecule lying on it!) I tried to run a MR with a model with two alternative positions and adjusted occupancy for the third molecule, but the Rfactor/free get higher ( 40%) and the map becomes worse – even the good ones (molecules 1 and 2) and for third molecule it remains bad (or worse). A procedure that “solved” the problem (decreased the Rfactor/free and gave good maps for third molecule) was the following: I integrated and scaled the data in P21, then I ran the sfcheck and it showed a twinned data (probably because of the (pseudo) higher symmetry present – P21212). So, I detwinned the data (with detwinn) and run a MR with detwinned data that gave a very good solution with tree molecules in ASU (it have never happened before!). After the MR I refined this MR solution against the original P21 data (without detwinn procedure) with amplitude based twin refinement in Refmac5 and, finally, it gave a good statistics (R factor / free about 0.19 / 0.22; FOM ~0.8) in the first round of refinement. I think that procedure probably discard reflections related to other positions making increasing the signal of the most frequent position. Link (.pdf): https://dl.dropbox.com/u/16221126/superposition.pdf Is there some problem in procedure described? If so, does anybody have a suggestion how can I model these disorder? Moreover, it seems to be a long range disorder (multiples positions along the all lattice), since even in P1 the maps for this third molecule are very bad. Thank you for all the suggestions. Cheers, Andrey 2013/3/25 Eleanor Dodson eleanor.dod...@york.ac.uk First - I dont think you have a 3rd molecule where you have put it - or at least not one with full occupancy. Those maps are a clear indication that something is wrong. What is the Matthews coefficient for the numbers in the asymmetric unit? Presumably your processing gave you a lattice which fitted the diffraction spots? ie you didnt miss a set of observations? You should see that at the data processing stage, and the different integration programs also try to report it. If there is non-crystallographic translation that can confuse things a bit; some classes of reflections might be systematically weak, but you can find if there is such a phenomena by doing a patterson. Or run ctruncate after merging the data - it checks this, and so does Xtriage. All these options will also check for twinning. If there is NCT then that could explain the high Rfactor. Are the spots nicely shaped? There are some cases of sheared crystals, which usually show up in distorted diffraction spots. If this is so and you have integrated the data according to an orthogonal lattice, there is nothing to stop you merging those observations in a low symmetry. Pointless gives you good statistics on the scoring for different symmetry operators. You can either run MR again in that symmetry - check all SGS consistent with the pointgroup, or try to work out how to position your P22121 solution in the new SG. There may well be 2n+1 copies of your molecule when you double the size of the asymmetric unit - all hard to check without more information. Good luck Eleanor On 22 March 2013 17:54, Andrey Nascimento andreynascime...@gmail.comwrote: Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the
Re: [ccp4bb] delete subject
That last paragraph is great: Adam is the author of the book Surviving Your Stupid, Stupid Decision to Go to Grad School (Broadway Books, 2010) -- Bill On Mar 28, 2013, at 9:09 AM, Ian Tickle ianj...@gmail.com wrote: By coincidence this just landed in my Inbox: http://membercentral.aaas.org/multimedia/webinars/how-recruit-citizen-scientists-discovery So maybe after all Tom is way ahead of the rest of us in his structure-solving strategy - though I agree with others that his tactics need to be honed somewhat! Cheers -- Ian On 28 March 2013 14:43, Raji Edayathumangalam r...@brandeis.edu wrote: Ed, I very much agree with you. We've all had to learn that questions posted to ccp4bb and the ensuing discussions take on a life of their own. Once one posts a question on ccp4bb, there's no such thing as steering the direction of the discussion on the ccp4bb and there's no such thing as the equivalent of screaming Stop! Stop! Stop! on the ccp4bb. Also, I don't believe people simply woke up one day and posted irritating or mean comments to ccp4bb. Ed was spot on for why some folks reacted the way they did to the post so let's acknowledge that as well. I didn't get the impression that any of the replies suggested that students stop posting questions. There are many many students on this BB who are in small institutions without even the minimal help at arm's length and who get tons of help from posting questions to the ccp4bb. That situation is not all that distant in my own memory and I suspect for many other experts on this BB. But posting 10MB attachments and getting the entire ccp4bb community to crowdsource towards problem solving is all good, but only to a certain degree. It may be great to get things done quickly with the collective intellect of the ccp4bb but there comes a point when the correct answers may get fed back at such a rapid speed that if one doesn't go back and try to figure stuff out for oneself, including the reasons/theory/logic behind the answers/solutions that the community has posted, it may be to the detriment of one's own learning, especially if one is in the early stages of learning the subject matter. Cheers, Raji On Thu, Mar 28, 2013 at 9:09 AM, Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2013-03-28 at 12:15 +, Tom Van den Bergh wrote: I think this is a good time to end the discussion. As a general comment, discussions on boards like ccp4bb often digress and take direction different from you original intent. I may understand your desire to try to control the situation, but if people on this board feel that the questions of data sharing, student training, netiquette and proper choice of resolution cutoff are worthy of further discussion (that may not have much to do with specifics of your original request for assistance), it is their right too. What may have caused some extra grief is this unfortunate turn of phrase in your original post Could you try some refinement for me, because this is first structure that i need to solve as a student and i dont have too many experience with it. It goes a bit beyond the usual my R-values are too high what should I do question and may be instinctively construed as if you expect someone to actually do your work for you (I am sure that is not what you asked). So a bit of a vigorous reaction that you received likely results from misunderstanding your intent (albeit posting your data is very unusual and strengthens the impression) and perhaps misplaced feeling that you have abandoned attempts to resolve the problem independently too soon. I did *not* look at your data and therefore I may be completely wrong here, but it is my understanding that your actual issue was not realizing there could be more than one molecule in the asymmetric unit. More traditional route is to describe your situation in general terms and offer to provide data to those willing to take a closer look. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] delete subject
Hi all, This has indeed been a highly informative and educational thread from many view points, and it highlights the opportunities and challenges that scientists face today by having access to tools like the CCP4BB . I just wanted to touch on something that was briefly alluded to at the early stages of this saga, and it has to do with data confidentiality and to some degree understanding the various policies that your institution adheres to. I raise this issue for the benefit of students like Tom, who may not have been exposed to the various implications that this brings. In my view, understanding your institution's (or your lab's) data sharing policies is extremely important prior to taking such action. In some institutions and specially in industry, sharing data without prior approval would be grounds for dismissal or even worst (lawsuits come to mind). So as we all learn from Tom's experience in this thread, I think we should all use good judgment when seeking help and deciding when to share data to an open forum. My 2 cents. Francisco From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji Edayathumangalam [r...@brandeis.edu] Sent: Thursday, March 28, 2013 7:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] delete subject Ed, I very much agree with you. We've all had to learn that questions posted to ccp4bb and the ensuing discussions take on a life of their own. Once one posts a question on ccp4bb, there's no such thing as steering the direction of the discussion on the ccp4bb and there's no such thing as the equivalent of screaming Stop! Stop! Stop! on the ccp4bb. Also, I don't believe people simply woke up one day and posted irritating or mean comments to ccp4bb. Ed was spot on for why some folks reacted the way they did to the post so let's acknowledge that as well. I didn't get the impression that any of the replies suggested that students stop posting questions. There are many many students on this BB who are in small institutions without even the minimal help at arm's length and who get tons of help from posting questions to the ccp4bb. That situation is not all that distant in my own memory and I suspect for many other experts on this BB. But posting 10MB attachments and getting the entire ccp4bb community to crowdsource towards problem solving is all good, but only to a certain degree. It may be great to get things done quickly with the collective intellect of the ccp4bb but there comes a point when the correct answers may get fed back at such a rapid speed that if one doesn't go back and try to figure stuff out for oneself, including the reasons/theory/logic behind the answers/solutions that the community has posted, it may be to the detriment of one's own learning, especially if one is in the early stages of learning the subject matter. Cheers, Raji On Thu, Mar 28, 2013 at 9:09 AM, Ed Pozharski epozh...@umaryland.edumailto:epozh...@umaryland.edu wrote: On Thu, 2013-03-28 at 12:15 +, Tom Van den Bergh wrote: I think this is a good time to end the discussion. As a general comment, discussions on boards like ccp4bb often digress and take direction different from you original intent. I may understand your desire to try to control the situation, but if people on this board feel that the questions of data sharing, student training, netiquette and proper choice of resolution cutoff are worthy of further discussion (that may not have much to do with specifics of your original request for assistance), it is their right too. What may have caused some extra grief is this unfortunate turn of phrase in your original post Could you try some refinement for me, because this is first structure that i need to solve as a student and i dont have too many experience with it. It goes a bit beyond the usual my R-values are too high what should I do question and may be instinctively construed as if you expect someone to actually do your work for you (I am sure that is not what you asked). So a bit of a vigorous reaction that you received likely results from misunderstanding your intent (albeit posting your data is very unusual and strengthens the impression) and perhaps misplaced feeling that you have abandoned attempts to resolve the problem independently too soon. I did *not* look at your data and therefore I may be completely wrong here, but it is my understanding that your actual issue was not realizing there could be more than one molecule in the asymmetric unit. More traditional route is to describe your situation in general terms and offer to provide data to those willing to take a closer look. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] delete subject
No. :-) When you are a reviewer for structural papers in journals (I do this work sometimes), and when you see an article that has (in this example) Tom's structure in it, but he and/or his mentor is not an author, then you call the editor and tell them you may have a problem. I realize that the case may not be closed with that statement because the manuscript could indeed be totally legitimate and genuine, but it would be a signal in my mind to watch for. A friend could not just run with the data and publish. A competing group could take advantage and get ahead in their project inexpensively (provided that the posted data are what you think they are). But that is sort of the point of publishing result (I must remember to leave my idealism at home tomorrow). Our old approach is to keep a lid on all your data until the paper is published. Although it is hard to imagine, there could be a mechanism by which you make all your data public, immediately when you get it and this public record shows who owns it. The advantage (in my mind) of such a system would be that you would also make public the data that does not make sense to you (it does not fit your scientific model) and this could (and has) lead to great discoveries. The disadvantage to the method is that you will sometimes post experiments that are just completely wrong (you did not measure what you said you measured) and this might make you look dumb (not really, this happens all the time; a favorite saying is 'we all make mistakes, we just make sure they don't leave the room'). And furthermore, you would finally have a journal of unpublishable data, where all the experiments that we should not have done for one reason or another reside and can act as a warning what not to do in the future. It is possible that I am socialist. In the US that is not a good thing, but I don't worry about it. Furthermore, teaching/learning is a concern. More and more places no longer have the resources or the patience to teach or learn crystallography. I once heard a friend say something along these lines: people who did not learn crystallography are now teaching the next generation. As proof for that, he explained that experiments are done at synchrotrons that clearly show that not the beamline is broken, but the operator does not understand the concepts and therefore the data collected are not useful. In my world I see crystallography as a tool, and no longer as a goal all by itself (it was a goal when I was a graduate student). I am frequently concerned that protein crystallography will go the way of small molecule crystallography: a few places provide this service and as an experimentalist you don't much worry about how they do it. Of course, until it becomes super-easy to produce high-quality protein and crystals, this won't happen. Mark With apologies to Tom, I don't have a stop-button, Raji is right about that. -Original Message- From: vellieux frederic.velli...@ibs.fr To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Mar 28, 2013 1:54 am Subject: Re: [ccp4bb] delete subject Hello, I stayed away from this thread until now - the major reason being that I was fitting snugly under my quilt. However I feel compelled to react now: placing your data in a public repository (thereby proving that you did the work) also means that a colleague, friend or whatever can and will publish your work for you. Once your work has been published you cannot publish it again, you did the work and the colleague, friend or whatever has in fact appropriated your work. In the world of dreams I was living in until a few moments ago (it was night time), this is perhaps the way we should act. In the real world we live in, even your colleague upstairs will publish your work if he / she has a chance to do it because by doing so he / she will improve his / her career while ensuring that yours doesn't take off. Fred. On 28/03/13 01:34, mjvdwo...@netscape.net wrote: Earlier today, I thought this and didnot write it. It is a slightly different theme on your suggestion: I hear there are now (but have not seen examples of) journals (web sites) where you do exactlywhat Tom did: you put your data there, which proves that you did the work (first) and you do not worry about the fact that you are making it public before formal publication, because makingdata public is the reason why you got the datain the first place. And nobody can claim to havedone the work, because everybody knows thatsomeone else was first - the web site is
Re: [ccp4bb] delete subject
On Thu, Mar 28, 2013 at 11:28 AM, mjvdwo...@netscape.net wrote: Although it is hard to imagine, there could be a mechanism by which you make all your data public, immediately when you get it and this public record shows who owns it. http://deposit.rcsb.org (or international equivalent) The advantage (in my mind) of such a system would be that you would also make public the data that does not make sense to you (it does not fit your scientific model) and this could (and has) lead to great discoveries. The disadvantage to the method is that you will sometimes post experiments that are just completely wrong There is a further problem: since as Frank pointed out, structures are increasingly less valuable without accompanying non-crystallographic experiments, there is a risk of other groups taking advantage of the availability of data and performing the experiments that *you* had hoped to do. Or, similarly, a group who already has compelling biochemical data lacking a structural explanation would immediately have everything they needed to publish. Either way, you would be deprived of what might have been a thorough and genuinely novel publication. Since most employment and funding decisions in the academic world are made on the basis of original and high-profile research and not simply number of structures deposited in the PDB, this puts the crystallographer at a distinct disadvantage. This isn't purely hypothetical - a grad school classmate who worked on genome sequences complained about the same problem (in her case, the problem was bioinformatics groups analyzing the data - freely available on the NCBI site, as mandated by the funding agencies - before the sequencing was even complete). Of course the same argument has been used in the past against immediate release of PDB entries upon publication - and the community (quite appropriately, IMHO) rejected it as nonsense. I actually like the idea of releasing data ASAP without waiting to publish, but it has a lot of practical difficulties. -Nat
[ccp4bb] fplc system
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] delete subject
On 28/03/2013 18:50, Nat Echols wrote: http://deposit.rcsb.org (or international equivalent) The advantage (in my mind) of such a system would be that you would also make public the data that does not make sense to you (it does not fit your scientific model) and this could (and has) lead to great discoveries. The disadvantage to the method is that you will sometimes post experiments that are just completely wrong There is a further problem: since as Frank pointed out, structures are increasingly less valuable without accompanying non-crystallographic experiments, there is a risk of other groups taking advantage of the availability of data and performing the experiments that *you* had hoped to do. Or, similarly, a group who already has compelling biochemical data lacking a structural explanation would immediately have everything they needed to publish. Either way, you would be deprived of what might have been a thorough and genuinely novel publication. Since most employment and funding decisions in the academic world are made on the basis of original and high-profile research and not simply number of structures deposited in the PDB, this puts the crystallographer at a distinct disadvantage. If someone has already done the other experiments, the absolutely best outcome for society is for the two to get together and write the paper as co-authors -- instead of precious funding money being wasted with a second fool doing exactly the same experiments in a silly rat-race. Lovely, so that leaves us with the trivial question of making people acknowledge other people's data when they publish. I suppose we can ask Watson for pointers (not Crick, he's not around anymore).
Re: [ccp4bb] delete subject
Hey everyone, Both Mark and Fred make some good points. I totally agree with Nat (beat me to the send button). Although in an ideal world with all the advancements in crowd sourcing and electronic media, one might think that posting data on a bulletin board might be considered marking one's turf and protect the scientist place in that pathway towards discoveries. Regrettably, the current reality doesn't' support this case. As structural biologists, we are still in the mode of first to publish gets the bulk of the glory and potentially future funding on the topic. For instance, when I was in graduate school, the lab I was in had KcsA crystals at the same time as a couple of competing groups. Several groups including the one I belong to had initial diffraction data. One group was able to solve KcsA, the first K channel trans-membrane protein structure, first. That group was led by Roderick Mackinnon, now a Noble Laureate partly because of this work. Now imagine if one of Mackinnon's student would have put up the web their initial diffraction data and another group would have used it to assist in their interpretation of their own data and either solved the structure before Mackinnon, or at least published it prior. Even if they acknowledged Mackinnion for the assistance of his data (as they should), Mackinnion and the other scientists in his lab would likely not have received the broad acclaim that they received and justly deserved. Also, ask Rosalind Franklin how data sharing worked out for her. Times haven't changed that much since ~10 years ago. Actually, as many have mentioned, things have potentially gotten worse. Worse in the respect that the scientific impact of structure is increasingly largely tide to the biochemical/biological studies that accompany the structure. In other words, the discoveries based on the insights the structure provides. Understandably, this increasing emphasis on follow up experiments to get into high impact journals in many cases increases the time between solving the structure and publishing it. During this gap, the group who solved the structure first is vulnerable to being scoped. Once scoped unless the interpretation of the structure and the conclusion of the follow up experiments are largely and justifiably divergent from the initial publications, there is usually a significant difficulty getting the article published in a top tier journal. Many might argue that they deposited it first, but I haven't seen anyone win that argument either. Because follow up articles will cite the publication describing the structure, not the PDB entry. Naturally, many could and should argue that this isn't they way it should be. We could rapidly move science ahead in many cases if research groups were entirely transparent and made available their discovers as soon as they could meet the veracity of peer-review. However, this is not the current reality or model we operate in. So, until this changes, one might be cautious about tipping your competition off whether they be another structural biology group looking to publish their already solved structure, or biology group that could use insights gathered by your structure information for a publication that might limit your own ability to publish. Fortunately, for Tom his structure sounds like it is only important to a pretty specific scientific question that many folks might not be working on exactly. Scott On Thu, Mar 28, 2013 at 12:28 PM, mjvdwo...@netscape.net wrote: No. :-) When you are a reviewer for structural papers in journals (I do this work sometimes), and when you see an article that has (in this example) Tom's structure in it, but he and/or his mentor is not an author, then you call the editor and tell them you may have a problem. I realize that the casemay not be closed with that statement because the manuscript could indeed be totally legitimate and genuine, but it would be a signal in my mind to watch for. A friend could not just run with the data and publish. A competing group could take advantage and get ahead in their project inexpensively (provided that the posted data are what you think they are). But that is sort of the point of publishing result (I must remember to leave my idealism at home tomorrow). Our old approach is to keep a lid on all your data until the paper is published. Although it is hard to imagine, there could be a mechanism by which you make all your data public, immediately when you get it and this public record shows who owns it. The advantage (in my mind) of such a system would be that you would also make public the data that does not make sense to you (it does not fit your scientific model) and this could (and has) lead to great discoveries. The disadvantage to the method is that you will sometimes post experiments that are just completely wrong (you did not measure what you said you measured) and this might make you look dumb (not really, this
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] delete subject
Ideally, one would be engaged in a collaboration wherein structure can be linked to function and published together. For most of us, that indeed would require a collaboration, since resources for most of us (at least in the US at present) are scant. Few crystallographers have the ability and resources to follow-up on the structure with mutagenesis or other data by themselves. For me, mammalian cell culture and transfection experiments are not feasible. Of course, in some cases, a structure can “explain” existing biological/biochemical data. These are the “primo” projects in which the structure by itself is valuable enough to merit publication in a top-tier journal. This is great when you can find it. However, those cases are becoming increasingly rare. One undercurrent in this discussion is this: a structure by itself is not enough to make it to the top tier journals in most cases. So what are we to do? My belief is that we have to share the credit and accept the fact that sometimes we will be secondary authors on major publications where the biology really counts. I guess I would rather be part of a team effort like that, in which I am not the first or last author, when the work is of high value, than publish my structure in a specialty journal. In a really competitive field, the choice can be difficult without collaborators. Yet, over time, even secondary authorship on high-profile projects, can accumulate justified scientific praise. At least I hope so. With diminishing resources, what choice do we really have? Dave On 3/28/13 5:06 PM, Scott Pegan scott.d.pe...@gmail.com wrote: Hey everyone, Both Mark and Fred make some good points. I totally agree with Nat (beat me to the send button). Although in an ideal world with all the advancements in crowd sourcing and electronic media, one might think that posting data on a bulletin board might be considered marking one's turf and protect the scientist place in that pathway towards discoveries. Regrettably, the current reality doesn't' support this case. As structural biologists, we are still in the mode of first to publish gets the bulk of the glory and potentially future funding on the topic. For instance, when I was in graduate school, the lab I was in had KcsA crystals at the same time as a couple of competing groups. Several groups including the one I belong to had initial diffraction data. One group was able to solve KcsA, the first K channel trans-membrane protein structure, first. That group was led by Roderick Mackinnon, now a Noble Laureate partly because of this work. Now imagine if one of Mackinnon's student would have put up the web their initial diffraction data and another group would have used it to assist in their interpretation of their own data and either solved the structure before Mackinnon, or at least published it prior. Even if they acknowledged Mackinnion for the assistance of his data (as they should), Mackinnion and the other scientists in his lab would likely not have received the broad acclaim that they received and justly deserved. Also, ask Rosalind Franklin how data sharing worked out for her. Times haven't changed that much since ~10 years ago. Actually, as many have mentioned, things have potentially gotten worse. Worse in the respect that the scientific impact of structure is increasingly largely tide to the biochemical/biological studies that accompany the structure. In other words, the discoveries based on the insights the structure provides. Understandably, this increasing emphasis on follow up experiments to get into high impact journals in many cases increases the time between solving the structure and publishing it. During this gap, the group who solved the structure first is vulnerable to being scoped. Once scoped unless the interpretation of the structure and the conclusion of the follow up experiments are largely and justifiably divergent from the initial publications, there is usually a significant difficulty getting the article published in a top tier journal. Many might argue that they deposited it first, but I haven't seen anyone win that argument either. Because follow up articles will cite the publication describing the structure, not the PDB entry. Naturally, many could and should argue that this isn't they way it should be. We could rapidly move science ahead in many cases if research groups were entirely transparent and made available their discovers as soon as they could meet the veracity of peer-review. However, this is not the current reality or model we operate in. So, until this changes, one might be cautious about tipping your competition off whether they be another structural biology group looking to publish their already solved structure, or biology group that could use insights gathered by your structure information for a publication that might limit your own ability to publish. Fortunately, for Tom his
[ccp4bb] lcp crystal extraction
Hi All, I am wondering if anyone has tried to retrieve a crystal from a Molecular Dimension LCP plate. What would you recommend as a method? The plastic is too flexible to be cut as it squashes the drop when you try it. Is there another way someone has successfully done this? Thanks in advance for your input. Konstantin
Re: [ccp4bb] lcp crystal extraction
Hi Konstantin, I use #11 Classic Fine Point Blades from X-acto to cut through the plastic Laminex film covers without much of an issue. If you decide to use glass covers rather than plastic, Hampton sells a nice glass-cutting tool ( http://hamptonresearch.com/product_detail.aspx?cid=18sid=203pid=626). Cheers, Jim On Thu, Mar 28, 2013 at 3:26 PM, Konstantin Knoblich knobl...@wehi.edu.auwrote: Hi All, I am wondering if anyone has tried to retrieve a crystal from a Molecular Dimension LCP plate. What would you recommend as a method? The plastic is too flexible to be cut as it squashes the drop when you try it. Is there another way someone has successfully done this? Thanks in advance for your input. Konstantin -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
[ccp4bb] best transient mammalian expression systems..?
Dear all, I would be interested in comments/opinions about what is your experience on which would be the best mammalian cell culture vector/cell line/medium combination for transient expression to get the best yields. Depends, of course, but anyway obviously there are differences, HEKs are suppose to be easy to transfect, but which flavour and how and what medium or would you prefer CHO? (probably more so for stable cell lines?) ...etc. vectors...? Or does it make a difference (it seems to). Good reviews/comparisons? is it worth going beyond DMEM and supplements? (what do you add in?) Stupid questions - trying to educate myself a bit here.. and i am willing to spend a bit or at least know what difference it makes (time is money anyway ...and worse). and we are not doing suspension at the moment, but could consider it if it seems like a good idea. Thanks for the tips.., Tommi
Re: [ccp4bb] Hydrophobic contacts
Hi Kavya, My guess is that you are referring to the van der Waals (vdw) contribution of the hydrophobic effect (the other being of entropic nature). If this is the case, then the most reasonable consensus is that Dij Ri + Rj + 2 R(H2O), where Dij is the distance between atoms i and j, Ri is the vdw radius of atom i, Rj, the vdw radius of atom j and R(H2O) the vdw radius of the water molecule. Doing so, you state that as far as two atoms are close enough to avoid being solvated (either one!), then they are close enough to engage in vdw interaction and if the atoms being considered are both nonpolar, that would correspond to what you call hydrophobic interaction. Therefore, the distances that you state are likely meaningless, depending on the sizes of the atoms concerned. I suppose you are using unified atoms; in which case, e.g., CH2 is about 1.8 Ang. If you take R(H2O) = 1.4 Ang, then 1.8 + 1.8 + 2x1.4 = 5.0 Ang. 8 Ang is far too much for CH2/CH2! HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet at univ-lorraine.fr LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. - Mail original - Dear users, Sorry for an off-topic question. What is the limits for hydrophobic interactions in protein? Some prefer 5Ang some prefer upto 8Ang. Any reference or suggestions are welcome. Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Hydrophobic contacts
The best reference by far for the hydrophobic interaction is Israelachvili's Intermolecular and Surface Forces. Anyone really interested in the topic will probably also like books by Ninham such as Hyde et al, The Language of Shape. I cannot really recommend the later book Ninham and Lo Nostro, Molecular forces and self-assembly because it is too disorganised, but there is some interesting material in there. The classics by Tanford are always worth a read too: The Hydrophobic Effect Ben Franklin stilled the waves. (A personal favourite source for student lectures). Hydrophobic effects can extend very far into solution - see Israelachvili for examples between surfaces extending to tens of nanometres. It really depends on your particular problem, and flat things tend to be stickier. Be aware that reducing analysis of hydrophobicity to a problem of Euclidean geometry, based on a static model with continuous solvent, involves assumptions which are not always met in practice. On Mar 29, 2013, at 9:09 AM, Nadir Mrabet wrote: Hi Kavya, My guess is that you are referring to the van der Waals (vdw) contribution of the hydrophobic effect (the other being of entropic nature). If this is the case, then the most reasonable consensus is that Dij Ri + Rj + 2 R(H2O), where Dij is the distance between atoms i and j, Ri is the vdw radius of atom i, Rj, the vdw radius of atom j and R(H2O) the vdw radius of the water molecule. Doing so, you state that as far as two atoms are close enough to avoid being solvated (either one!), then they are close enough to engage in vdw interaction and if the atoms being considered are both nonpolar, that would correspond to what you call hydrophobic interaction. Therefore, the distances that you state are likely meaningless, depending on the sizes of the atoms concerned. I suppose you are using unified atoms; in which case, e.g., CH2 is about 1.8 Ang. If you take R(H2O) = 1.4 Ang, then 1.8 + 1.8 + 2x1.4 = 5.0 Ang. 8 Ang is far too much for CH2/CH2! HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet at univ-lorraine.fr LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. Dear users, Sorry for an off-topic question. What is the limits for hydrophobic interactions in protein? Some prefer 5Ang some prefer upto 8Ang. Any reference or suggestions are welcome. Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Hydrophobic contacts
Sorry, a bit late here (2:00 AM) after a long day. I meant (for CH2/CH2 contacts!): Dij 1.8 + 1.8 + 2x1.4 = 6.4 Ang. Best, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet at univ-lorraine.fr LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. - Mail original - Hi Kavya, My guess is that you are referring to the van der Waals (vdw) contribution of the hydrophobic effect (the other being of entropic nature). If this is the case, then the most reasonable consensus is that Dij Ri + Rj + 2 R(H2O), where Dij is the distance between atoms i and j, Ri is the vdw radius of atom i, Rj, the vdw radius of atom j and R(H2O) the vdw radius of the water molecule. Doing so, you state that as far as two atoms are close enough to avoid being solvated (either one!), then they are close enough to engage in vdw interaction and if the atoms being considered are both nonpolar, that would correspond to what you call hydrophobic interaction. Therefore, the distances that you state are likely meaningless, depending on the sizes of the atoms concerned. I suppose you are using unified atoms; in which case, e.g., CH2 is about 1.8 Ang. If you take R(H2O) = 1.4 Ang, then 1.8 + 1.8 + 2x1.4 = 5.0 Ang. 8 Ang is far too much for CH2/CH2! HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet at univ-lorraine.fr LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. - Mail original - Dear users, Sorry for an off-topic question. What is the limits for hydrophobic interactions in protein? Some prefer 5Ang some prefer upto 8Ang. Any reference or suggestions are welcome. Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] delete subject
Scott, I'm not sure I understand your last paragraph. Once researchers have had their data pass peer review (which I interpret as meaning a journal has accepted it), how often do you think it happens that it does not immediately get published? Just depositing data in the PDB, or posting it on a public web site, is not meet[ing] the veracity of peer review. There is something to be said for giving credit to the first people who have subjected their data to peer review and had the data pass that step, otherwise people will be tempted to just post data of dubious quality to stake a public claim before the quality of the data has been independently checked. In a case where this initial public non-peer-reviewed posting is of unacceptable data quality, that would dilute credit granted to another person who later obtained good data. An unfortunate number of problematic structures still sneak through peer review. Relaxing quality review standards that must be passed before a scientist gets to claim credit for a discovery is a step backwards IMO. Cheers, Eric On Mar 28, 2013, at 5:06 PM, Scott Pegan wrote: Hey everyone, Both Mark and Fred make some good points. I totally agree with Nat (beat me to the send button). Although in an ideal world with all the advancements in crowd sourcing and electronic media, one might think that posting data on a bulletin board might be considered marking one's turf and protect the scientist place in that pathway towards discoveries. Regrettably, the current reality doesn't' support this case. As structural biologists, we are still in the mode of first to publish gets the bulk of the glory and potentially future funding on the topic. For instance, when I was in graduate school, the lab I was in had KcsA crystals at the same time as a couple of competing groups. Several groups including the one I belong to had initial diffraction data. One group was able to solve KcsA, the first K channel trans-membrane protein structure, first. That group was led by Roderick Mackinnon, now a Noble Laureate partly because of this work. Now imagine if one of Mackinnon's student would have put up the web their initial diffraction data and another group would have used it to assist in their interpretation of their own data and either solved the structure before Mackinnon, or at least published it prior. Even if they acknowledged Mackinnion for the assistance of his data (as they should), Mackinnion and the other scientists in his lab would likely not have received the broad acclaim that they received and justly deserved. Also, ask Rosalind Franklin how data sharing worked out for her. Times haven't changed that much since ~10 years ago. Actually, as many have mentioned, things have potentially gotten worse. Worse in the respect that the scientific impact of structure is increasingly largely tide to the biochemical/biological studies that accompany the structure. In other words, the discoveries based on the insights the structure provides. Understandably, this increasing emphasis on follow up experiments to get into high impact journals in many cases increases the time between solving the structure and publishing it. During this gap, the group who solved the structure first is vulnerable to being scoped. Once scoped unless the interpretation of the structure and the conclusion of the follow up experiments are largely and justifiably divergent from the initial publications, there is usually a significant difficulty getting the article published in a top tier journal. Many might argue that they deposited it first, but I haven't seen anyone win that argument either. Because follow up articles will cite the publication describing the structure, not the PDB entry. Naturally, many could and should argue that this isn't they way it should be. We could rapidly move science ahead in many cases if research groups were entirely transparent and made available their discovers as soon as they could meet the veracity of peer-review. However, this is not the current reality or model we operate in. So, until this changes, one might be cautious about tipping your competition off whether they be another structural biology group looking to publish their already solved structure, or biology group that could use insights gathered by your structure information for a publication that might limit your own ability to publish. Fortunately, for Tom his structure sounds like it is only important to a pretty specific scientific question that many folks might not be working on exactly. Scott
