Re: [ccp4bb] phaser: high z score but no sol
Hi Lisa, Why are you so sure there are 4 molecules in the ASU? There may only be 3 and forcing a fourth molecule is causing lots of clashes. In a similar case, I have seen phaser put two molecules right on top of each other when I forced it to search for too many molecules. In your case I would look at the solution (crystal packing) in coot and see whether the clashes are due to some loops (or domains) which may move upon ligand binding, or whether one of the molecules is at a completely wrong position and if you get a good crystal packing (crystal contacts in all directions) with fewer molecules. You can also start refinement with fewer molecules and see if additional density for missing molecule(s) appears. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of LISA Sent: Thursday, April 19, 2012 8:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phaser: high z score but no sol Hi all, I am trying to solve one structure by molecular replacement with phaser in CCP4. This a complex of a multi-domain domains with small ligand. I have structues of this protein in apo state and with other similar ligand. The space group of this crystal is P21. This crystal should have 4 molecules in ASU. I used the full protein as model but did get any sol and LLG is below zero. Then each domain were used as the search models in phaser with rotation and tranlsation. I can the get high z score (20), and LLG is raising. It looks like I get the right sol, but it have more 50 clashes. Why phaser give wrong sol with so high z socre? Can anyone give me some suggestion to solve my strucutes? Thank you. Best Lisa
Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
(my last spam) This is very true. Compared to biomedicine, protein crystallographers are holy saints: Of 50 landmark papers in oncology, people from Amgen could only reproduce 6 (11%) and in a similar study, people at Bayer could only reproduce 14 out of 67 (21%) studies. Even more troubling, non-reproducible papers got cited more often then reproducible ones. I really hope the bubble will collapse soon since it led (leads) to the waste of billions of research euros (in industry and academia) and the testing of ineffective compounds on patients. Sorry for this off-topic remark, Herman http://www.nature.com/nature/journal/v483/n7391/full/483531a.html http://www.nature.com/nrd/journal/v10/n9/full/nrd3439-c1.html -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Miguel Ortiz Lombardia Sent: Thursday, April 19, 2012 9:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers El 19/04/12 18:42, Patrick Loll escribió: Well, it is clear from this comment that in different fields there are different rules... . In macromolecular Xtallolgraphy, where some people deal with biologists from biomedical sciences, the impact of journals is an important aspect during evaluation and, unfortunately, pre-publication review of structures has no actual value in their field. For a structural BIO-logist in biomedical sciences, a paper it is not just a paper, it is an effort of years reduced to a (or few) paper(s). The non-structural BIO-people understand what is a Cell paper, but not at all about what it is a pre-publication of a structure. My thougts go in the direction of grant applications, fellowships, promotion, all filtered by the impact factor but not by pre-publication of structures which, btw, it is neither considered in the h-index of a researcher. Oh what the hell, someone else poured the gasoline, I may as well supply a lit match: What Maria says is absolutely true--I dwell among biologists, so I fully understand the rules of the field. But it's not so clear that these rules are good ones. Biology is obsessed with high impact, and I argue science is ill served by this preoccupation. The highest impact-factor journals seem to have the highest number of retractions (see this past Tuesday's New York Times Science section for a discussion). And in this forum it's certainly germane to note that the technical quality of published structures is, on average, poorer in the highest impact journals (at least by some criteria; see the paper from Brown Ramaswamy in Acta Crystallogr D63: 941-50 (2007)). Pat -- - Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu Indeed the rules are clearly bad. They're actually a mirror of the rules of political economy in our western/capitalist/call-them-as-you-want societies. Actually, expect bubble collapses in the biological field. Perhaps not spectacular, most probably not everything-falling-at-once, but surely not without serious implications. We also have our too-big-to-fall paradigms, especially in bio-medicine. In any case, the rules are there and for most of the people who intend to keep working (most often working as in job, not as in art) in biological science (with or without double quotes) it is certainly easier to bow to them than to resist them. Understandably, for the latter option is most often punished sooner or later, with no shame, by those who exclude you from the so-called excellence club. It would help if some big, truly respected names in biology would attack seriously these rules and put clear the damage they are causing to biological science. Some do, I'm now thinking of Peter Lawrence for example, but they are too few to be anything else than 'lone rangers'. It would be certainly even more helpful if we could unite and collectively reject this state of affairs. But this is, for several reasons that would need a far too-long text for a bulletin board post, less expected than rain on the desert. Whatever the case, we bio-crystallographers are a very small set of the people working in biology. We may now and then have this kind of discussion where we put forward our concerns, our idealistic view of the peer review system, etc. Move aside, go to a lab of almost any other field in biology and tell them about these discussions; most of the time they will look at you as they would at a Martian. Back to the original post: I have never been requested coordinates/data. It's however clear to me that if the reviewer wants to see them (s)he has the right to do so. The problem
[ccp4bb] three letter codes for buffer components
Hi, does anybody know if there is a published list (or does anybody have a list and is willing to share) with PDB three letter codes for typical buffer components that show up in crystal structures? Cheers Ruth -- Dr. Ruth Brenk Biological Chemistry and Drug Discovery College of Life Sciences University of Dundee Dow St. Dundee, DD1 5EH U.K. +44 (0)1382 386230 (phone) +44 (0)1382 386373 (fax) http://www.brenkgroup.dundee.ac.uk http://www.lifesci.dundee.ac.uk/people/ruth_brenk/ The University of Dundee is a registered Scottish charity, No: SC015096. The University of Dundee is a registered Scottish Charity, No: SC015096
Re: [ccp4bb] Molecular replacement
Thank you very much! Eleanor On 19 April 2012 19:43, Bret Wallace bretw...@gmail.com wrote: I noticed this missing when I first installed v. 6.2 as well. They noted this in the problems page. You just need to replace the phaser_MR.tcl file with the updated version in the updates page. I tried this earlier and it created a new line in the GUI to specify the packing criterion under Additional Parameters. I think this is what you were referring to? Bret
Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
When I hear of a reviewer holding up a publication and then publishing something similar, my first reaction is fury and I feel the case should be investigated and this immoral individual should be exposed. However I can see that there are many shades of gray here. We're all biased in that we tend to ignore information that conflicts with our previous cherished beliefs and focus on things that confirm them. So it can take a long time to change your mind - sometimes months. This can lead to indecision and delays, but in retrospect we tend to think that we would have come to those conclusions in any case so there's no harm in using the info. People with a strong sense of duty will get the review done quickly and make sure that they don't take advantage of the data, but I can see that it can be tempting. I think the idea of getting reviewers to sign a piece of paper saying that there is no immediate conflict of interest i.e. they are not about to publish something similar, is a good one. The author could prepare simple statement describing the topics covered (not the abstract which gives, or should give, the conclusions). Then it's not a matter of proving that the reviewer cheated, only that they had the opportunity to cheat. I always communicate freely with the editors, e.g. telling them why I don't want such-and-such to review the paper. Wouldn't it be possible simply to ask the editor to check that the reviewer asking for co-ordinates etc is not close to publishing something that could benefit from the data? I don't think it's a good idea for reviewers' names to be visible because that would mean that we would all have to do a far more professional job of the review. (I'm not a career scientist but I've been asked to review a few papers.) I also agree with those who say that this competitive focus on high impact journals etc. stifles creativity, is inefficient and gives poor value for money. Just some thoughts - probably stating the obvious Patrick On 20 April 2012 01:18, Edward A. Berry ber...@upstate.edu wrote: Bosch, Juergen wrote: To pick a bit on George's point with MR citation. Here's how you can read it in the paper from your favourite competitor: A homology model was generated using [fill in any program for ab initio prediction] and subsequently used for molecular replacement with Molrep. The structure was refined to an Rwork of 21% and Rfree of 24 %. Or maybe the structure was solved by MIR, using a lot of heavy atom data that they had been unable to solve until a fortuitous MR result gave phases which located the heavy atoms -- No, No, it was that new improved version of autosol! Anyway, who cares how the heavy atoms were located- the structure was solved entirely using their data and they have the raw data (image files even) to prove it. It was just bad luck with the derivatives that kept them from solving it 6 months earlier. They really deserve to have the first publication! -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] PhD position - Institut de Biologie Structurale (Grenoble)
A PhD position funded by a grant is available in the Synchrotron Group headed by Dr Jean-Luc Ferrer at Institut de Biologie Structurale (IBS http://www.ibs.fr/spip.php?lang=en) at Grenoble, starting in October 2012. The project will focus on the structural and biochemical characterizations of proteins from plant and protein interactions. The successful candidate will be employed for a period of three years, with a gross salary of around 1700 EUR/month. We search for candidates holding a MSc degree in biochemistry. Experience in protein purification, and/or background in protein crystallography will be an advantage. IBS is close to the European large instruments, the ILL http://www.ill.eu/ and the ESRF http://www.esrf.eu/. Please send CV and 2 reference letters to david.cobe...@ibs.fr and jean-luc.fer...@ibs.fr -- David Cobessi Institut de Biologie Structurale 41, Rue Jules Horowitz 38027 Grenoble Cedex-1, France Tel:33(0)438789613 33(0)608164340 Fax:33(0)438785122
[ccp4bb] new XDS version
Dear XDS users, for about 3 weeks a new (non-commercial) version has been available from http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds/ . It incorporates improvements, bug fixes, and new features. If there are any problems/regressions with a new XDS version, let me (or Wolfgang Kabsch) know; in such a case pls consider sharing the data (confidentially) with us that allow to reproduce the problem. If you wish to be notified of future new versions, please send me an email and I'll add you to my list. thanks, Kay -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature smime.p7s. smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
Just a thought: When a reviewer asks for the model/data, 1) The reviewer should be given at most 24-48 hours of time to give comments after receiving the data. 2) (S)he should declare to the editor that the paper is going to be accepted if everything with the data/model is okay. The reviewer should also send comments to author on what does (s)he intend to examine in the structure. 3) After going through the model/data, the reviewer's comment should be exclusively based on the structure or its correlation with the experimental data. 4) If reviewer finds any mistake which can not be corrected or which changes the theme of the paper and the reviewer rejects the paper, the responsibility should lie on author. But certainly the editor or a team decided by editor should ensure that when the paper is rejected at this stage, the reason for rejection is valid and the mistakes can not be rectified. Editor should also ensure that authors are given sufficient opportunity to correct the mistake if possible. Thanks MT On Fri, Apr 20, 2012 at 6:23 AM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: When I hear of a reviewer holding up a publication and then publishing something similar, my first reaction is fury and I feel the case should be investigated and this immoral individual should be exposed. However I can see that there are many shades of gray here. We're all biased in that we tend to ignore information that conflicts with our previous cherished beliefs and focus on things that confirm them. So it can take a long time to change your mind - sometimes months. This can lead to indecision and delays, but in retrospect we tend to think that we would have come to those conclusions in any case so there's no harm in using the info. People with a strong sense of duty will get the review done quickly and make sure that they don't take advantage of the data, but I can see that it can be tempting. I think the idea of getting reviewers to sign a piece of paper saying that there is no immediate conflict of interest i.e. they are not about to publish something similar, is a good one. The author could prepare simple statement describing the topics covered (not the abstract which gives, or should give, the conclusions). Then it's not a matter of proving that the reviewer cheated, only that they had the opportunity to cheat. I always communicate freely with the editors, e.g. telling them why I don't want such-and-such to review the paper. Wouldn't it be possible simply to ask the editor to check that the reviewer asking for co-ordinates etc is not close to publishing something that could benefit from the data? I don't think it's a good idea for reviewers' names to be visible because that would mean that we would all have to do a far more professional job of the review. (I'm not a career scientist but I've been asked to review a few papers.) I also agree with those who say that this competitive focus on high impact journals etc. stifles creativity, is inefficient and gives poor value for money. Just some thoughts - probably stating the obvious Patrick On 20 April 2012 01:18, Edward A. Berry ber...@upstate.edu wrote: Bosch, Juergen wrote: To pick a bit on George's point with MR citation. Here's how you can read it in the paper from your favourite competitor: A homology model was generated using [fill in any program for ab initio prediction] and subsequently used for molecular replacement with Molrep. The structure was refined to an Rwork of 21% and Rfree of 24 %. Or maybe the structure was solved by MIR, using a lot of heavy atom data that they had been unable to solve until a fortuitous MR result gave phases which located the heavy atoms -- No, No, it was that new improved version of autosol! Anyway, who cares how the heavy atoms were located- the structure was solved entirely using their data and they have the raw data (image files even) to prove it. It was just bad luck with the derivatives that kept them from solving it 6 months earlier. They really deserve to have the first publication! -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Off-topic: Site-directed mutagenesis
Theresa, For point mutations, we currently use MEGAWHOP, which is a megaprimer-based whole plasmid PCR method. It has the advantage of using single mutant primers of modest length (21-24 nt) in combination with existing flanking primers for the target gene (either the 5' or 3' flanking primers). We used to do a two-step megaprimer PCR to copy out the whole mutant gene but don't bother anymore and just do a one-step mutant megaprimer before doing the MEGAWHOP. The final product requires DpnI digestion in-situ prior to transformation of E. coli. Our protocol can be found at: http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Recombinant+DNA+Protocolssaved_msg=y#Site_directed_mutagenesis_by_Megaprimer_Quick_Change_MEGAWHOP_PCR It's not elegant but it works. We'll have to try PIPE sometime. Looks like it saves some time over MEGAWHOP at the expense of designing two primers for each variant gene. I guess it depends if time is more important than money or money is more important than time. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 4/17/2012 5:53 PM, Theresa Hsu wrote: Dear all I would like to get some opinions on site-directed mutagenesis. What are the current methods available? I know the Quick Change, are there others that work better? Thank you.
Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
It seems that this discussion has somehow reached the conclusion that if a reviewer asks for model/data, there absolutely must be an ulterior motive to cheat you out of your high profile publication. On the other hand, it seems like the intent of such reviewer is also misunderstood as if the only reason would be to catch you fabricating data. I dare to suggest that neither is correct and while this discussion seems to have developed along these lines, both only represent a small fraction of real life situations. I routinely request unreleased data/models. I do it to stem the tide of subprime models in the PDB (outright fabrication is very very rare) and it helps me to form judgment on presented model interpretation (which is more difficult/often impossible to do from 2D figures). If an author refuses to provide data, I would refuse to review. Don't mind my name disclosed in exchange for data, secrecy is for totalitarian governments. Cheers, Ed. On Thu, 2012-04-19 at 20:18 -0400, Edward A. Berry wrote: Bosch, Juergen wrote: To pick a bit on George's point with MR citation. Here's how you can read it in the paper from your favourite competitor: A homology model was generated using [fill in any program for ab initio prediction] and subsequently used for molecular replacement with Molrep. The structure was refined to an Rwork of 21% and Rfree of 24 %. Or maybe the structure was solved by MIR, using a lot of heavy atom data that they had been unable to solve until a fortuitous MR result gave phases which located the heavy atoms -- No, No, it was that new improved version of autosol! Anyway, who cares how the heavy atoms were located- the structure was solved entirely using their data and they have the raw data (image files even) to prove it. It was just bad luck with the derivatives that kept them from solving it 6 months earlier. They really deserve to have the first publication! -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] phaser: high z score but no sol
Would the self rotation map make sense with 4 molecules ? Jürgen Sent from my iPad On Apr 20, 2012, at 2:55, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: Hi Lisa, Why are you so sure there are 4 molecules in the ASU? There may only be 3 and forcing a fourth molecule is causing lots of clashes. In a similar case, I have seen phaser put two molecules right on top of each other when I forced it to search for too many molecules. In your case I would look at the solution (crystal packing) in coot and see whether the clashes are due to some loops (or domains) which may move upon ligand binding, or whether one of the molecules is at a completely wrong position and if you get a good crystal packing (crystal contacts in all directions) with fewer molecules. You can also start refinement with fewer molecules and see if additional density for missing molecule(s) appears. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of LISA Sent: Thursday, April 19, 2012 8:20 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phaser: high z score but no sol Hi all, I am trying to solve one structure by molecular replacement with phaser in CCP4. This a complex of a multi-domain domains with small ligand. I have structues of this protein in apo state and with other similar ligand. The space group of this crystal is P21. This crystal should have 4 molecules in ASU. I used the full protein as model but did get any sol and LLG is below zero. Then each domain were used as the search models in phaser with rotation and tranlsation. I can the get high z score (20), and LLG is raising. It looks like I get the right sol, but it have more 50 clashes. Why phaser give wrong sol with so high z socre? Can anyone give me some suggestion to solve my strucutes? Thank you. Best Lisa
Re: [ccp4bb] phaser: high z score but no sol
P21.. You sure about this space group? (very high confidences for space group and laue group in pointless?) F On Apr 19, 2012, at 12:20 AM, LISA wrote: Hi all, I am trying to solve one structure by molecular replacement with phaser in CCP4. This a complex of a multi-domain domains with small ligand. I have structues of this protein in apo state and with other similar ligand. The space group of this crystal is P21. This crystal should have 4 molecules in ASU. I used the full protein as model but did get any sol and LLG is below zero. Then each domain were used as the search models in phaser with rotation and tranlsation. I can the get high z score (20), and LLG is raising. It looks like I get the right sol, but it have more 50 clashes. Why phaser give wrong sol with so high z socre? Can anyone give me some suggestion to solve my strucutes? Thank you. Best Lisa
