[ccp4bb] EMBL Australia Group Leader Positions; Monash University, Melbourne
Hi All We are seeking applicants for two EMBL Australia Group leaders to be based at Monash University in Melbourne, Australia: We seek future research leaders in Electron Microscopy (EM) and in protein crystallography. In the protein crystallography field we particularly encourage researchers with outstanding track records in working with challenging membrane protein systems to apply. The new recruits will form a key part of the Biomedical Discovery Institute being developed within the Faculty of Biomedical and Psychological Science. In the EM field we are interested in recruiting scientists with expertise in correlative light and electron microscopy (CLEM), high-resolution electron tomography or in determining protein structures at high-resolution using EM. The successful applicants will be expected to develop a multidisciplinary, collaborative and innovative research program that addresses highly significant biological problems. Exciting opportunities for collaboration with chemists, immunologists and physicists (including X-ray free electron laser science) will be provided through the ARC Centre of Excellence in Advanced Molecular Imaging (www.imagingcoe.org/). Further significant interactions are anticipated with GPCR and Ion channels focused researchers within the Monash Institute of Pharmaceutical Science. Successful applicants will have access to excellent light and electron microscopy platforms (including an FEI Titan Krios equipped with a Falcon 2 detector), protein production and protein crystallography facilities (including robotics dedicated for experiments involving lipidic cubic phases). Monash University adjoins the Australian Synchrotron, which houses beamlines dedicated to micro-focus crystallography and Small Angle X-ray Scattering experiments. (see also advert Nature / Science http://www.embl.de/aboutus/jobs/partners/download/EMBL-Australia-GLs.pdf) Please email (in English) a cover letter clearly stating position(s) being applied for, your CV, names and contact details of 3 referees, and a summary of current and future research interests to: positi...@emblaustralia.org. Applications close 2nd November, 2014. Interviews will be held 18-20th February 2015. Cheers James -- Professor James Whisstock Director of the ARC Centre of Excellence for Advanced Molecular Imaging NHMRC Senior Principal Research Fellow Department of Biochemistry and Molecular Biology Monash University Clayton Campus Melbourne VIC 3800 Australia Phone: +61 418 170 585 Website: www.imagingcoe.org Twitter: @imagingCoe *Join us at the 3rd Prato conference on Pore Forming Proteins, Prato, Italy - 12th to 15th May, 2015: www.pores2015.org http://www.pores2015.org/ *
Re: [ccp4bb] very informative - Trends in Data Fabrication
As famously observed by the Yes Minister teamthe dream outcome for any organization: http://www.youtube.com/watch?v=x-5zEb1oS9Afeature=youtube_gdata_player J Sent from my iPhone On 07/04/2012, at 3:16 AM, Patrick Loll pat.l...@drexel.edu wrote: Ron makes an excellent point. Many institutions devote far more energy to limiting risk than to doing the right thing. This leads administrators to a frightening, but logical conclusion: The less science we do, the less chance of our doing something that could invite a penalty on the university. This translates into rules intended to head off bad behavior, but which in fact make it more difficult to do honest science, and increase the administrative burden (our IT group has already made great strides in this direction--if you can't connect to the network, then you can't use it to violate HIPAA!). So I agree that we should be cautious about improvements. Pat On 6 Apr 2012, at 12:23 PM, Ronald E Stenkamp wrote: Dear John, Your points are well taken and they're consistent with policies and practices in the US as well. I wonder about the nature of the employer's responsibility though. I sit on some university committees, and the impression I get is that much of the time, the employers are interested in reducing their legal liabilities, not protecting the integrity of science. The end result is the same though in that the employers get involved and oversee the handling of scientific misconduct. What is unclear to me is whether the system for dealing with misconduct is broken. It seems to work pretty well from my viewpoint. No system is perfect for identifying fraud, errors, etc, and I understand the idea that improvements might be possible. However, too many improvements might break the system as well. Ron --- 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
Re: [ccp4bb] very informative - Trends in Data Fabrication
Hi I was thinking about the last statement in the Acta editorial - It is important to note, however, that in neither of these cases was a single frame of data collected. Not one.. This brought me back to the images.. To date there is no global acceptance that original diffractiom images must be deposited (though I personally think there should be). Many of the arguments around this issue relate to the time and space required to house such data. However (and apologies if this has already been raised and I have missed it), if our sole intent is to ascertain that there's no trouble at t'mill then deposition of a modest wedge of data and / or a 0 and 90, while not ideal, may be sufficient to provide a decent additional check and balance, particularly if such images, headers etc were automatically analysed as part of the already excellent validation tools in development. I'm sure there are a number of clever ways (that could be unadvertised or kept confidential to the pdb) that could be used to check off sufficient variables within such data such that it should (?) be very difficult to falsify images without triggering alarm bells. Of course this would probably then drive those that are truly bonkers to attempt to fabricate realistically noisy false diffraction images, however I would hope that such a scheme might make things just a little more difficult for those with fraudulent intent, particularly if no one (apart from the developers) knows precisely how and what the checking software checks! While it seems sad that it's come to this cell biologists and biochemists have had to deal with more and more sophisticated versions of the photoshopped western for years. Accordingly, most high profile journals run figures through commercial software that does a reasonable job of detection of such issues. J Sent from my iPhone On 03/04/2012, at 11:10 PM, Dyda d...@ulti.niddk.nih.gov wrote: I think that to review a paper containing a structure derived from crystallographic data should indeed involve the referee having access to coordinates and to the electron density. Without this access it is not possible to judge the quality and very often even the soundness of statements in the paper. I think the argument that this may give a competitive advantage to the referee who him or herself maybe working on the same thing should be mute, as I thought article refereeing was supposed to be a confidential process. Breaching this would be a serious ethical violation. In my experience, before agreeing to review, we see the abstract, I was always thought that I was supposed to decline if there is a potential conflict with my own work. Perhaps naively, but I always assumed that everyone acts like this. Unfortunately however, there is another serious issue. After a very troubling experience with a paper I reviewed, I discussed this issue with journal editors. What they said was that they already have a hell of time to find people who agree to referee, by raising the task level (asking refs to look at coords and density) they feared that no one would agree. Actually, perhaps many have noticed the large number of 5 liner referee reports saying really not much about a full length research article. People simply don't have the time to put the effort in. So I am not sure how realistic is to ask even more, for something that at some level, is pro bono work. Fred [32m*** Fred Dyda, Ph.D. Phone:301-402-4496 Laboratory of Molecular BiologyFax: 301-496-0201 DHHS/NIH/NIDDK e-mail:fred.d...@nih.gov Bldg. 5. Room 303 Bethesda, MD 20892-0560 URGENT message e-mail: 2022476...@mms.att.net Google maps coords: 39.000597, -77.102102 http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred ***[m
[ccp4bb] Postdoctoral position at Monash University
Hi all Position posting on behalf of Rob Pike. Please direct all enquires to rob.p...@monash.edu. Prof. Robert Pike is seeking a talented postdoctoral research fellow to work on protease structure and function in the Department of Biochemistry and Molecular Biology, Monash University. The successful candidate will have a recently awarded PhD and a background in protein crystallography, protein expression and purification. Some enzymology experience would also be highly desirable. The Monash Protein crystallography unit includes state of the art protein production and crystallization robotics together with excellent in-house data collection facilities. The University is furthermore situated next door to the Australian synchrotron, a national facility that includes two dedicated crystallography beamlines. -- Professor James Whisstock ARC Federation Fellow Department of Biochemistry and Molecular Biology Monash University Clayton Campus Melbourne VIC 3800 Australia Phone: +61 418 170 585
Re: [ccp4bb] off-topic: Discovery Studio software
Hi What specific features are you looking for? Pymol (www.pymol.org) has most standard tools for analysis of protein structure as well as some very basic modeling capacity. Evaluation and academic packages are available. J On Saturday, 19 February 2011, Padmaja Mehta-D'souza padmaja-mehta-dso...@omrf.org wrote: I have a few questions about Discovery Studio (DS): -Will it work on a Mac OS?-Is there another program (free or cheaper) that will do everything DS does?-How much better is DS compared to the old InsightII program?-Any other comments from users/naysayers about DS Thanks! Padmaja Padmaja Mehta Research Assistant Member and Director, Biacore Core Facility, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
[ccp4bb] Two Research fellowship positions
Hi all We are seeking one or two postdoctoral researchers with a demonstrated expertise in biochemistry and protein crystallography and an interest in the structure and function of pore forming proteins and membrane proteins. Each fellowship includes a consumables budget as well as funding for the fellow to support a PhD stipend in their laboratory. The fellowships are funded through the Australian Research Council Super Science Fellowships program, the aim of which is to attract and retain outstanding early-career researchers and help them to transition to an independent research career in Australia. The fellowships are each three year positions and will be based at Monash University in Melbourne, Australia. Successful candidates will have an exceptional track record including significant published work. Please note that these ARC super science research fellowships are generally restricted to scientists who have been awarded their PhD in the past three years. Applications should be sent to elaine.pear...@monash.edu and should include a cover letter, a one page executive summary and a full CV. Applications close 2nd September 2010. See: http://monash.turborecruit.com.au/job/job_details.cfm?id=503700 Cheers James Whisstock Enquiries about these positions should be sent to: james.whisst...@monash.edu -- Professor James Whisstock ARC Federation Fellow Honorary NHMRC Principal Research Fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, Building 77, VIC, 3800, Australia +613 9902 9312 (Phone) +613 9902 9500 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] Retraction of 12 structures and experimental data deposition
Dear Felix I agree - to address this Ash Buckle and colleagues have set up TARDIS (http://tardis.edu.au/experiment/view/) and built the associated tools for relatively painless deposition of data for registered users. As well as making the data available to others we find that this is also a great way of cleanly and permanently archiving the raw data! Furthermore, it seems that University libraries are keen to support electronic data deposition and archiving (not just Xtal data) in general, since their role in storing and providing access to paper journals is somewhat diminished! J Felix Frolow mbfro...@post.tau.ac.il wrote: In mathematics, when one is making a claim of solving the longstanding mathematical problem, it is a tradition that his colleagues mathematician will take care to check his solution. This solution MUST stood up to the scrutiny of the world's expert. As an example see http://en.wikipedia.org/wiki/Andrew_Wiles. Two papers of Wiles and WilesTaylor's were published in Annals of Mathematics( both Nature and Science do not publish mathematical papers probably due to the fact that mathematicians were outcasted by Alfred Noble due to the unknown reason) and were checked by international community. It is pity, that when in MX society the similar claim/claims is/are made, we do not have an access to all information needed to put this claim to scrutiny as the policy of experimental data deposition is not very clear. It would be convenient to have/to give an access to the data (not necessarily raw) such as native and derivatives hkl, I, sigI files, all with separated Friedel pairs and log files in PDF format of the scaling programs, preferably in graphical form such as Report of HKL2000. This is needed for the community to check and for desired for educational purposes if one would like to reproduce the way of structure determination of a difficult problem. I personally have now idea what will be discovered in some most famous cases, and probably as a result young structural biologists will be reinforced in their believe that the structures could be derived from the most marginal phase information such as in the case of http://www.nature.com/nature/journal/v426/n6967/full/nature02200.html Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Dec 12, 2009, at 24:27 , Ed Pozharski wrote: I would like to point out that this outright fabrication remains an isolated incident. There are over 50,000 crystal structures in the PDB, which means that this is only ~0.02% of the total. This is all quite bad, but let's not overstate the problem. Maybe such report is not a great idea after all - don't you think it will help the next troublemaker to fabricate structures better? :) I wonder if there are any structures from the author in question that were not fabricated. After all, some serious mistakes were made in 2hr0 (how can you not know about bulk solvent and B-factor variation?). There is 1BGX which was not on retraction list (I guess since it was done at Temple University, not UAB), but it looks weird too: the B-factors of main chain and side chain atoms are not much different from each other and this 2.3A-diffracting crystal has very few crystal contacts (it's the Taq DNA polymerase in complex with inhibitory Fab and the whole heavy chain makes no crystal contacts at all which is probably unique). On Fri, 2009-12-11 at 15:30 -0500, Ibrahim Moustafa wrote: You are absolutely right, more information describing to what extents these structures were falsified will be valuable to the community. Actually, it will be more useful if the investigators can publish their report as an article in Acta D (as a case study for tracking falsified structures). I have a suggestion (actually a request) to the expertise in the field to write a kind of review article about sources of error in crystallography and how to hunt these errors. It will be even better if it is written considering the non-crystallographers (scientists who use the structural information - like the co-authors on structural papers). This will help to educate the non-crystallographers how to look at the structures critically. Ibrahim -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse / -- Professor James Whisstock ARC
Re: [ccp4bb] images
Hi Sean Ash Buckle has already developed one! Tools for general deposition will be released shortly. http://tardis.edu.au/ Cheers J Sean Seaver s...@p212121.com wrote: I started a poll to find out whether crystallographers need and are interested in an X-ray diffraction data bank. Will crystallographers find this resource helpful and be willing to submit their structures? I hope you will take a moment to share your opinion via the poll and/or by posting any questions or comments you may have. I will follow up with results in about a month. http://www.p212121.com/2009/06/04/do-we-need-an-x-ray-diffraction-image-data-bank/ Thanks. Sean Seaver -- Professor James Whisstock ARC Federation Fellow Honorary NHMRC Principal Research Fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] buster-tnt on OSX ?
I heartily agree with Ash and Gerald - Autosharp (which we routinely use for experimental phasing) is free for academics and in my opinion is an outstanding package. Further, we have always found the people at Global Phasing are always extremely helpful with all sorts of phasing questions not just installation! From a personal perspective I would always rather have free or low cost packages that require a little bit of setup than being charged like a wounded bull for a commercial package. J Ashley Buckle [EMAIL PROTECTED] wrote: Having solved a few structures using AutoSHARP, I feel compelled to comment on the CCP4BB that I really like the software, never had a major problem installing or using it, feel extremely grateful to the guys at Global Phasing for making this available to us all, and for (in my case at least) responding to my queries quickly and helpfully. cheers Ashley On 26/10/2008, at 7:40 PM, James Stroud wrote: The info at http://www.globalphasing.com/buster/installation/index.html#requirements will give you some hints about whether it will be successful on OS X. As per the Global Phasing modus operandi, any instruction involving installation of their software is muddled in riddle and ambiguity. They have made an art of making their software difficult to install. I guess the philosophy is that if you can somehow get their software installed, then you have earned your phase information. Personally, I'd rather solve my structure with using chisanbop and a pencil than attempt to install Global Phasing software. Speaking from experience, this is my 2c. Apologies if I hurt anyone's feelings who are just trying to make a buck. James On Oct 24, 2008, at 9:39 AM, jacques-philippe colletier wrote: Hi everydoby, I`d like to know if there is a version of BUSTER-TNT available on MacOSX ? Anyone knows ? * Dr. Jacques-Philippe Colletier UCLA / DOE Institute for Genomics and Proteomics 90095 Los Angeles, CA, USA * [EMAIL PROTECTED] [EMAIL PROTECTED] * -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com Associate Professor Ashley M Buckle NHMRC Senior Research Fellow The Department of Biochemistry and Molecular Biology School of Biomedical Sciences, Faculty of Medicine Victorian Bioinformatics Consortium (VBC) Monash University, Clayton, Vic 3800 Australia http://www.med.monash.edu.au/biochem/staff/abuckle.html iChat/AIM: blindcaptaincat skype: ashley.buckle Tel: (613) 9902 0269 (office) Tel: (613) 9905 1653 (lab) Fax : (613) 9905 4699 -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] skin on a drop
Hi Try setting up at 4C can help J Hi All, As we know, there is a skin on a crystallization drop, especially when the drop contains PEG. Does anyone know what the skin is, a degraded protein ? How to prevent it ? Thanks Simon
Re: [ccp4bb] MR with 40% identical model failed
Hi Have you checked for twinning? J TC Hu [EMAIL PROTECTED] wrote: Dear all, I am working on a structure which is composed of two domains. The X-ray data is quite good (P21, 2.2A, Rmerge=0.08, 93% completeness), there is one molecule (277 residues) per ASU. The structure has four homologous structures with sequence identities ranging from 35%-40%. The four structures superposed quite well and there is no relative movement between the two domains. However, MR with these probes all failed using Molrep or Phaser (R/Rfree after refinement is 51%/55%, and the density is poor). Then I searched the two domains separately and located the first one (about 60% of the structure, 44% identity) unambiguously but failed to find the second one (36% identity) either by searching it alone or fixing domain 1. The R and Rfree after refining domain 1 are 39% and 44%. The density is good for the existing model but disconnected in the missing part of the structure in which I can hardly place any residue. Only an obscure shape of beta sheet could be recognized but its position relative to domain 1 is quite different from those in the homologous structures. Thus I suspect that domain 2 has undergone movement or adopted a distinctive conformation comparing to the known structures. I searched the CCP4BB archive and found a similar case from Eric Liu posted in the end of July this year. The suggestions are to mask the whole molecule and perform DM, or to carry out OASIS-DM-Building iterative cycles (as published in Acta Cryst. (2007). D63, 793-799), or to measure the experimental phases, and etc. So I tried to make a solvent mask in coot 0.3.3 using the mask map by molecule function. I read in the map (from refining the first barrel) and the superposed complete homologous structure, masked the map by it, and exported the masked map. Then I used MAPMASK utility to convert the map to mask (the density cutoff is set to 1) and feed it to DM. But the resulted MTZ contained all zero reflections. Thus my questions are: 1) How can I make a mask as mentioned above? Am I doing it the wrong way? 2) Is there any other method to improve the density of the missing structure? I tried several MR probes of the second barrel fixing the first one but all failed. Sorry for the naive question, but this has bothered me for quite some time. Any suggestions will be greatly appreciated. Thanks in advance! Tiancen Hu Shanghai Institute of Materia Medica Chinese Academy of Sciences -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] R-sleep
Maybe we could invent an R_Schrodinger that hovers in a quantum state untill we peek :) J William Scott [EMAIL PROTECTED] wrote: If R-sleep is to be the real validation R-factor, why not just sequester each of R-sleep and the current R-free, each as a randomly-chosen (but mutually exclusive) set of reflections, and then proceed as normally with the other (eg) 80% of the data until the very end of the refinement, using the R-free set to optimize weightings for geometries, NCS symmetry averaging, and so forth, and then simply add those back in at the penultimate step of refinement. In the end, you have R-sleep and the Rfactor corresponding to the rest of the data, just like before, and you can have the additional statistic reporting the difference between R-sleep and and R-free, which we could call something like the R-i-didn't-peak. Peter Adrian Meyer wrote: This raises a slightly tangential question though - how do we know how what obs/param ratio is good enough? -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] Quick soak method
Hi - sorry - rather than iodine I meant to say we had had success with Potassium Iodide (1M for 20 seconds)! Cheers James [EMAIL PROTECTED] wrote: Hi, I do not use their method as such - however, I love heavy atom soaks and do them any time I can, so I've got very similar experiences in the past. Heavy atoms can bind very quickly even from quite dilute solutions - the quickest I've ever soaked (and got useful data) was sodium chloroplatinate in under ten minutes at ~ 1mM concentration. The next quickest was K2Pt(NO2)4 which tends to take between 10 minutes and 30 minutes, almost regardless of the concentration. This is by far my favorite HAD reagent, incidentally. In general my soaks are all less than one hour with a few exceptions, such as iodine [not iodide!] soak to iodinate tyrosines which is typically better done during a day or so with very low amount of I2, via vapor phase. Artem Hi, I'd like to find out how successful the quick soak method for heavy atom derivatisation proposed by Radaev and Sun: Sun PD, Radaev S, Kattah M. Generating isomorphous heavy-atom derivatives by a quick-soak method. Part I: test cases. Acta Cryst. 2002. D58:1092-1098. has been in comparison to the classical method of longer soaks at low concentrations of heavy atom compound. The method was quite successful in our hands a few years ago but (fortunately?) it's becoming increasingly rare that we use heavy atoms. I understand that evidence will necessarily be anecdotal, but let's not let that stop us. Derek -- Derek Logan tel: +46 46 222 1443 Associate professor fax: +46 46 222 4692 Molecular Biophysicsmob: +46 76 8585 707 Lund University Box 124, Lund, Sweden -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] worst mr probes
Hi Bill Scott used a RNA fragment based MR approach in his paper on Ribozyme structure. The Structural Basis of Ribozyme-Catalyzed RNA Assembly Michael P. Robertson and William G. Scott Science 16 March 2007: Vol. 315. no. 5818, pp. 1549 - 1553 In our lab we had an interesting time with the two forms of Glutamate Decarboxylase. This is probably not as extreme as some of the stories posted, but may be useful. Initially we crystallised GAD65, at 2.3A resolution with 1 Mol in the ASU and C2221 with the two molecules in the dimer related by crystallographic symmetry. The MR probe (Dopa Decarboxylase) was about 17% identical and comprised ~33% of the final GAD structure (essentially part of the PLP domain). While we were able to get a clear MR solution using PHASER (correctness confirmed by the packing of the crystallographic dimer, which is physiologically relevant), we were unable to get over model bias. To address this we first tried heavy atom soaks to attempt phased MR, but always ran into serious non-isomorphous issues with the crystals. Since GAD has to be produced in yeast, MAD was not really an option. As a last resort, we crystallised GAD67 (~75% identical to GAD65) again crystals diffracted to 2.3A resolution but this time SG P21 with two molecules in the dimer related by NCS. This time, using a similar MR probe, we again got a clear solution, and in refinement NCS averaging won the day and we were to complete the structure. Its worth noting that we had a pretty unpleasant time trying a LOT of different MR models (at least 15-20) even for GAD67 and we had to progress very carefully with the building. Once GAD67 was solved, however, phasing, building and refinement of GAD65 was straightforward (as expected), using a GAD67 monomer as an MR probe. The work is described in: Fenalti et al GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nat Struct Mol Biol. 2007 Apr;14(4):280-6. Epub 2007 Mar 25 In the end, we were happy since comparison of the two structures was what we wanted to do. However, if only we had gone for GAD67 first, we would have saved a lot of pain!!! Cheers James Bryan W. Lepore [EMAIL PROTECTED] wrote: a quick summary on 'the worst MR probes' Pierre Rizkallah: X-ray Structure Solution of Amaryllis Lectin by Molecular Replacement with Only 4% of the Total Diffracting Matter. L. Chantalat, S.D. Wood, P.J. Rizkallah, C.D. Reynolds (1996). Acta Crystallographica, D52, pp. 1146-1152 Poul Nissen: Nissen P, Kjeldgaard M, Thirup S, Polekhina G, Reshetnikova L, Clark BF, Nyborg J (1995). Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science, 270, 1464-72. Anderson, et. al. (from Manfred Weiss) acta cryst d 52, 469-480 other pubs worth checking out: Schwarzenbacher, et. al. acta cryst d 60 1229 2004 Adams, et. al. acta cryst d 55, 181, 1999 all phaser refs. i also got a report of the correct phaser solution with Z-score=5.2/LLG=49 - for the uninitiated, a phaser Z-score of 5 to 6 is an 'unlikely' solution. any other references, stats appreciated thanks. -bryan -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] MS for verification of protein constructs
Hiya For verification purposes we almost always N-terminal sequence (we are fortunate in that we have such a facility on site so that the samples can be turned round fast) - old technology but good and solid! We then usually combine these data with a Mass Spectra (MALDI-ToF-Tof - on site - is usually accurate enough to see 1-2 amino acid difference) to check for C-terminal trimming, PTMs / other issues. We usually perform these experiments both on purified material and crystals run on a gel, particularly if Captain Paranoid has paid me a visit and we're always if we are about to embark or MIR or MAD etc(someone once told me a horror story of battling for years to purify a protein and solve a structure only to find it was a lysozome contaminant - probably an urban myth but scary enough)_ J Anastassis Perrakis [EMAIL PROTECTED] wrote: ... all these are correct indeed - but ESI-TOF is also a nice solution, especially coupled to an LC system. My understanding was that MALDI-TOF is better for smaller fragments, accuracy can be about 10 Dalton ... for more info there is a useful short review of the use of ms techniques for structural work in: http://journals.iucr.org/d/issues/2006/10/00/gx5084/index.html (the 'spine' issue). A. On 5 Sep 2007, at 22:56, Joel Guenther wrote: If you have a very pure protein sample, you'll want to use an ESI- ion trap for analyzing proteins of that size. It should be possible to get an exact mass (i.e. within a single Da). It's possible, but very rare, to get exact masses of proteins up to 100 kDa using ESI-ion trap instruments. If your sample is not highly purified, you'll need to use some type of TOF instrument. MALDI-TOF should work. With a TOF instrument, you shouldn't expect to be able to distinguish between point mutants of a protein, but you'll be able to get information about larger changes. For instance, you'll be able to determine if you completely cleaved a his-tag off a construct. Our lab is spoiled because we have access to the HHMI Mass Spec facility, but I'd imagine that there are many facilities that can get accurate masses on 40 kDa proteins. -Joel = Joel M. Guenther PhD Candidate, Department of Chemistry Kuriyan Laboratory http://jkweb.berkeley.edu/ University of California, Berkeley 176 Stanley Hall, QB3 Berkeley, CA 94720-3220 tel: (510) 643 0166 fax: (510) 643 2352 = On 9/5/07, Jacob Keller [EMAIL PROTECTED] wrote: I second Dr. Loll's question, and would like to be CC'd in whatever MS tips, including service-providers, are sent. I have been having a bit of a debacle with a certain MS service provider. Jacob Keller ==Original message text=== On Wed, 05 Sep 2007 11:41:52 am CDT Patrick Loll wrote: I wonder if anyone would care to share experiences/ideas/biases that relate to the use of mass spectrometry to verify the identity of protein constructs used for crystallization. Our experience with different MS facilities has been checquered. Specifically: What's the current thinking on the best approach to get masses for intact proteins of moderate size (say, 40 kD)? ESI-TOF? What kind of resolution should one hope to obtain in such cases (10E-04?) Any suggestions as to good facilities offering fee for service MS characterization are welcome (but should be shared off line, I think; continental US only). Thanks, Pat -- -- --- Patrick J. Loll, Ph. D. (215) 762-7706 Associate Professor FAX: (215) 762-4452 Department 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 [EMAIL PROTECTED] ===End of original message text=== *** Jacob Keller Northwestern University 6541 N. Francisco #3 Chicago IL 60645 (847)491-2438 [EMAIL PROTECTED] *** -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] Off Topic:crystallization in the presence of glycerol
We have crystallised many things with 10% Glycerol. If % is high enough, there is also often the added bonus in that the crystals are naturally cryoprotected! J Edward Berry [EMAIL PROTECTED] wrote: We've grown crystals of the cytochrome bc1 complex in the presence of glycerol. I think as high as 25% in the initial droplet (protein in 50% glycerol mixed with equal volume of precipitant), but that was diluted somewhat by reverse vapor diffusion. Glycerol tends to increase the solubility in our case, so the PEG concentration we had to use in those experiments was higher than in later glycerol-free setups. Ed Rob Gruninger wrote: Dear All, I am working with a protein that requires 10% glycerol throughout the purification to keep it soluble. I have been very worried that having glycerol in my protein solution when I am trying to crystallize it will prevent me from obtaining crystals. I am curious to see if others have successfully obtained crystals of proteins that required glycerol to keep them soluble and if so what concentration of glycerol could you use? Should I be concerned about this or am I worrying too much? Thanks for your input Rob -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] crystal with precipitation
Sorry - this may have been mentioned previously, but have you tried banging in some glycerol (5-10%)? J shivesh kumar [EMAIL PROTECTED] wrote: Dear all I welcome all the suggestions regarding my crystals which is coming with the precipitation.The pI of the protein is 4.2 and the drop precipitates with MPD as low as 30% within 4-5 hrs.I am trying the pH ranging from 3.6-5.2 at 16C.There is no precipitation at 20% of MPD.Also,at high conc,the protein precipitates.The crystals are coming as florets as I mentioned with 2+2.Should I go for higher pH like uptp 8.5 or so.crystals are not growing bigger in size so that I can mount.Should I try changing the temperature.Ican use 4C and 16C.Right now I am keeping plates at 16C.I am concentrating the protein through lyophilization.As soon I pool it for Gel filtration,majority of it get precipitated,not soluble protein.Is there any method to make the protein stable? I appreciate all the informative suggestions.Thanx in advance and thanx everybody who has suggested the various options. Shivesh kumar -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] Data collection of SAD (MAD) data of Copper-containing protein
Hi For your SAD / MAD data, firstly after merging can you see any peaks in the anomolous difference patterson map - this is critical. You could try using SOLVE / RESOLVE or SHARP / AUTOSHARP to identify sites and to calculate phases, as you have a little bit more control over the process. You don't mention you Space group - are there any ambiguities here? If you have an poor initial MR model, together with some poor anomolous phases then you could use SHARP for phased molecular replacement. For PHASER we have found that (unsurprisingly) in low identity cases it can be very sensitive to the quality of the MR model - have you carefully pared down the model, removed loops, truncated non identical positions to C-beta etc? Its a little unclear from your mail (I may have missed some of this thread though) how many mols you think there are in the ASU - my advice is to start simple - ask PHASER to find one molecule (hit the all possible SG flag) and see what the LLG and Z-score is, if you get a significant hit, then fix and re-run asking it to find another (or just rerun asking it to find two) and so on - again LLG and Z-score are critical here. You may have to play with the clashing factor as sometimes good hits are rejected because of clashes between bits of the MR model that should have been deleted (if you consistently see logical packing but two bits of the structure clash that could be reasonably shifted then adjust your MR model accordingly) ! At each stage inspect the maps, the scores and the packing really carefully. If you have more than one template structures try an ensemble of models as well. Cheers J Yi Xue [EMAIL PROTECTED] wrote: So far, the native crystals diffracted best to 2.4A. The MAD data diffracted to 2.6~2.7A. We attempted to use phenix.hyss to identify copper atoms, and the program had hard time to identify the sites. The protein: Cu ratio is around 1:1, which is decided by ICP-AES measurement of the crystallization sample, not derived from the number of peaks in the anomalous map. The crystal contains copper as a cofactor, not a soaking derivative thing. As to oligomerization, it could be dimer or trimer, however, we don't have a model for it, since we dont know exactly, how does the drug link the monomers. I used phaser to do the MR, basically, what I did was to search one copy each time, the top 20 solutions will be used to start the search of the next copy. It stuck after finding four copies, and, I tried to change the some paramters for searching, such as percentage for peaks, it did not help. I would love the hear from experienced people about some tricks using phaser to solve some difficult MR cases. thanks a lot Yi -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] process SeMet labelled data
Dear Shivesh Below I've tried to give you some broad ideas about where to look / read and some packages / approaches to try. For a start there is a very comprehensive tutorial on the ccp4 site together with the experiemental phasing roadmaps. http://www.ccp4.ac.uk/dist/examples/tutorial/html/heavy-tutorial-mad.html which goes through the process step by step. For indexing and processing images, you are probably using MOSFLM or HKL2000?, both of which have excellent manuals that help with troubleshooting. At this stage you need to pick the appropriate bravais lattice then you need to refine and integrate. Once you have integrated (run in ccp4 for MOSFLM), then you need to scale the data using, for example, SCALA. Have a look at the following tutorial for running scala. http://www.ccp4.ac.uk/courses/ECM2004/runningscala.pdf Don't forget to seperate anomolous pairs when running scala Here, if relevant for your bravais lattice, you can also start to look at systematic absences to try to assign / narrow down the options for your space group. Remember, if your space group is incorrect, then (even though you may have a derivative) you won't be able to solve your structure. Thus if there are ambiguities, you need to systemically try out the options. Once you have scaled your data you may merge etc (see tutorial) using CAD and SCALEIT and start looking at some Anomalous Difference Patterson Maps to identify Heavy atom sites. see http://www.doe-mbi.ucla.edu/~sawaya/tutorials/Phasing/anopat.html Also, have a look at using SHARP/autoSHARP http://www.globalphasing.com/sharp/manual/index.html or SOLVE/RESOLVE http://www.solve.lanl.gov/ to analyse your data. These are both excellent packages for processing MAD / SAD datasets and calculating heavy atom positions / phases. For SHARP / autoSHARP you can supply the individual unmerged mtz's. SHARP will also alert you in a good humoured fashion to any deficiencies in your data and will try to pick the best approach (for e.g. it may try SAD depending on your data quality etc). autoSHARP will also run solvent flattening, give you final flattened maps readable in coot (which you should inspect to see if you can see features such as helices / strands etc). autoSHARP will even input your data into ARP/wARP, and if you are really fortunate you might even come in in the morning (watching ARP chain tracing can be rather addictive so its really best to go home!) and find that ARP has done a good job at a preliminary model or give you a nice starting point for manual building! Also for lower resolution and where you can clearly see features in the map for preliminary chain tracing try using Bucanneer. Hope this helps, good luck and above all have fun! Cheers James Dirk Kostrewa [EMAIL PROTECTED] wrote: Hi Mark, although Shivesh's question was not very specific, and he should have clearly given some more informations about what he would like to know, he is probably a beginner in crystallography and simply asked for help on this board. Not everyone has always time or is always in the mood to answer such questions. In my opinion, it's then better not to respond at all than to give an answer like yours that is neither helpful nor funny! We all should try to keep a good style here. Dirk. Mark J. van Raaij wrote: why don't you just send all your images to the ccp4bb, then we'll process them, solve the structure and publish it for you. And we might put you in the acknowledgements, if you are lucky. Mark On 28 Feb 2007, at 16:35, Jonathan Grimes wrote: Anastassis Perrakis wrote: On Feb 28, 2007, at 14:37, shivesh kumar wrote: Dear all, I have a data set at 2.2A, of the selenomethionene labelled protein.How should I process the data. Carefully ! Thanx for the help. Shivesh Tassos i am sure what tassos really meant was Very Carefully ! jon -- Dr. Jonathan M. Grimes, Royal Society Research FellowUniversity Research Lecturer Division of Structural Biology Wellcome Trust Centre for Human Genetics University of Oxford Roosevelt Drive, Oxford OX3 7BN, UK Email: [EMAIL PROTECTED], Web: www.strubi.ox.ac.uk Tel: (+44) - 1865 - 287561, FAX: (+44) - 1865 - 287547 Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia and Unidad de Rayos X, Edificio CACTUS Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ -- Dirk Kostrewa Paul Scherrer Institut Biomolecular Research, OFLC/110 CH-5232 Villigen PSI, Switzerland Phone:+41-56-310-4722 Fax: +41-56-310-5288 E-mail: [EMAIL PROTECTED] http://sb.web.psi.ch -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia
Re: [ccp4bb] software to calculate VDW interactions between small molecule and protein
HI - try the program contact from the ccp4 suite. http://www.ccp4.ac.uk/html/contact.html J Eleanor Dodson [EMAIL PROTECTED] wrote: mathias wrote: Dear all, Can anyone of you guys recommend free software, or any open access internet server, to calculate VDW interactions of small molecules binding to protein. The only information I need is an output file which lists all amino acids of the target protein which make VDW interactions with the binding small molecule. Thank you very much for your help and recommendations, Mathias Have you tried the MSD Pisa server at the EBI ? It lists all sorts of information? Eleanor -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
