[ccp4bb] Query - inhibitor screening by fluorescence based assay.
Dear All, Sorry for the non crystallography related question We are performing a fluorescence based assay to screen for inhibitor compounds of our enzyme and ultimately crystallize the enzyme along with inhibitor. We see that some of our compounds are autofluorescent and thus are effecting fluorescence. In such a case we make a compound blank (assay buffer+ compound) and subtract it from test (enzyme + compound) but still we see the values of test much higher compared to control (only enzyme). In this case can we bring down the values of compound balnk and test by a factor which brings the value of compound blank to the level of blank and compare the values of resultant test value with that of control after subtracting the respective blanks. For example we have the following arbitrary fluorescent readings (AFU) for one compound: Blank (70342) Control (163243) Compound balnk (1865830) Test (2020455) Since compound blank / blank ie (1865830)/(70342) = 26.52 can we do a normalization of compound blank and test values by a factor 26.52, which brings teh value of compound blank to teh level of blank and get the values (1865830)/26.52 = 70355 and 2020455/26.52 = 76186 and say the compound is inhibitor by comparing this test value with the value of control after subtracting teh values of respective blank. Thank you in advance, Suresh -- Suresh V Mattegunta MS Senior Research Associate Discovery Biology Division Aptuit Laurus PVT LTD. ICICI Knowledge Park Hyderabad 500078 India
Re: [ccp4bb] different compilers for ccp4 code
Charles Ballard schrieb: Hi Francois configure linux_intel_compilers should do the trick. We distribute ccp4 built with the intel compilers for OS X. Part of this is that the macs cover a much smaller range of cpus than linux boxes, so optimisation is less of a problem. If you want speed make sure that you are compiling for your architecture. My feeling, and I haven't checked this for some time, is that the intel compilers are a bit faster, 5-10% max, but that the gnu compilers have been closing the gap. true A pointed out by the previous post the intel compilers basically have an assert that goes if CPU=MD then stop. A number of years ago somebody published how to remove this from the code, and more recently intel lost a restrictive practice law case over it. So hopefully it will go away. That gives the wrong impression, I'm afraid. Intel mentions AMD processors in their Release Notes and compiler documentation. Please check out http://software.intel.com/en-us/articles/intel-fortran-compiler-for-linux-supported-operating-systems/ http://software.intel.com/en-us/articles/intel-fortran-compiler-intel-ia-32-processor-targeting-options/ and most importantly, http://software.intel.com/en-us/articles/performance-tools-for-software-developers-sse-generation-and-processor-specific-optimizations-continue/ In short, Intel compilers do not optimize specifically for AMD CPUs, but they do for Intel CPUs. As the manufacturer knows the instruction set of each CPU best, they are hardly to blame for that. There are compiler benchmarks at http://www.polyhedron.com/pb05-linux-f90bench_AMD0html - that gives you the information about how fast ist ifort on AMD, compared to other compilers. HTH, Kay Charles Ballard CCP4 ps- a lot of ccp4 is highly io dependent, so fast disks with decent cache can make a lot of difference On 18 May 2010, at 01:58, Francois Berenger wrote: Hello, This is not what you asked for, but I think it is good to know: Intel compilers don't seem to like non-Intel CPUs: http://www.agner.org/optimize/blog/read.php?iI Regards, F. Terry Lang wrote: Hey Everyone, I am considering switching from gcc to the Intel compiler in the hopes of making some of calculations run a bit faster. Has anyone ever tried compiling the ccp4 code base with the Intel compilers? Is there a difference in speed? What about in the reproducibility of the calculations? Any changes in statistics? Any information would be greatly appreciated! Thanks, Terry -- 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] different compilers for ccp4 code
On 19/05/10 09:40, Kay Diederichs wrote: Charles Ballard schrieb: Hi Francois configure linux_intel_compilers should do the trick. We distribute ccp4 built with the intel compilers for OS X. Part of this is that the macs cover a much smaller range of cpus than linux boxes, so optimisation is less of a problem. If you want speed make sure that you are compiling for your architecture. My feeling, and I haven't checked this for some time, is that the intel compilers are a bit faster, 5-10% max, but that the gnu compilers have been closing the gap. true It is extremely noticeable for cns. With similar optimizations you get a boost of more then 30% comparing the gcc/gfortran and ifc/icc builds. -- Justin Lecher Institute for Neuroscience and Biophysics ISB 3 - Institute for structural biochemistry Research Centre Juelich GmbH, 52425 Juelich,Germany phone: +49 2461 61 5385 signature.asc Description: OpenPGP digital signature
[ccp4bb] About seeding in microbatch under oil crystallization
Hi ccp4bbers, I want to know if anyone has any experience about seeding (streak seeding or microseeding) in microbatch under oil crystallization. I wonder if the oil might block or wipe away the seeds if cat whisker is used for streak seeding. Thank you for your input ahead. Best regards, Donghui
Re: [ccp4bb] Should I be worried about negative electron density?
Hi Jay, there have been a few discussions about sigma/rms levels, absolute values (e/A*3), significance levels etc on ccp4bb. See e.g. http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg06969.html http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg15273.html which are the ones I could find again (I'm sure there are others). One example: imagine you would know the true value of a distribution, say on 17 points. You then start to model it (this corresponds to your initial stages of refinement). After some cycles, you end up with a model you're happy with. It could look like this: point true start end - 11.0 1.1 1.00 21.1 1.2 1.10 31.5 1.4 1.52 42.0 2.3 2.01 52.7 2.9 2.72 63.0 3.5 3.02 74.0 4.2 4.04 86.0 6.3 6.04 99.010.0 9.09 106.0 6.3 6.04 114.0 4.2 4.04 123.0 3.5 3.02 132.7 2.9 2.73 142.0 2.3 2.01 151.5 1.4 1.52 161.1 1.2 1.10 171.0 1.1 1.00 Now plot those distributions (see attached file) and calculate mean/sigma of the differences (between the current model and the true values): start-true : mean =0.2471 with sd =0.2476 end-true : mean =0.0235 with sd =0.0219 You will see that point 9 always corresponds to a '3 sigma' difference peak - even if your model at the end is pretty much perfect (R-factor at the beginning is 9% and at the end down to 1%). Just because a distribution will always give you a mean and a sigma it doesn't mean that you need to chase after those '3 sigma' peaks forever. At least that is how I visualise the sigma/rms/electrons issue for me ... Cheers Clemens On Wed, May 19, 2010 at 12:13:54AM +0100, Jay Pan wrote: Hello Everyone, I have a reasonably well fitted electron density map through molecular replacement. However, there is always some red region left no matter how hard I tried when the mtz file is loaded into Coot. Is this because my model is still not good enough or it???s natural to most model fittings. In another word, should I be worried about the red region? Thanks in advance. Jay -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) *** attachment: ccp4bb_Fo-Fc.png
Re: [ccp4bb] About seeding in microbatch under oil crystallization
Hello Donghui we used a robot to setup the batch crystallization screens with some microseed stock solution and the relevant protein and reservoir stocks. That worked fine. I only mention a collegues work here, so if there are more detailed questions, I will approach him. Good luck, Harm On Wed, May 19, 2010 at 11:28 AM, wu donghui wdh0...@gmail.com wrote: Hi ccp4bbers, I want to know if anyone has any experience about seeding (streak seeding or microseeding) in microbatch under oil crystallization. I wonder if the oil might block or wipe away the seeds if cat whisker is used for streak seeding. Thank you for your input ahead. Best regards, Donghui -- Harm Otten Department of Chemistry Universitetsparken 5 2100 Copenhagen Ø Denmark # +45 35 32 02 89 fax +45 35 32 03 22 email h...@kemi.ku.dk Please consider the environment before printing this email.
Re: [ccp4bb] Should I be worried about negative electron density?
On 19 May 2010, at 00:36, Paul Emsley wrote: Jay Pan wrote: Hello Everyone, I have a reasonably well fitted electron density map through molecular replacement. However, there is always some red region left no matter how hard I tried when the mtz file is loaded into Coot. Is this because my model is still not good enough or it’s natural to most model fittings. In another word, should I be worried about the red region? Thanks in advance. Turn up the contour level and make it go away - that's what I do :) 3 or 3.5 sigma peaks are typical. Metals, carboxyls and disulfides are often associated with relatively strong negative density, some people try adjust their model to compensate (and others not, of course). As a rule of thumb, if you have 5 sigma peaks at the end of your refinement, that might be worrying/interesting. The median height of the tallest positive peak after autoBUSTER re-refinement for the PDB *depositions* during April this year is about 7.2, and of the tallest negative peak about -5.0. 25/50/75th quantiles: negative peaks -5.9 / -5.0 / -5.6 positive peaks 6.1 / 7.2 / 8.6 Tom Womack (Global Phasing)
Re: [ccp4bb] Native Gel Theory and Practice
Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] About seeding in microbatch under oil crystallization
Dear Patrick, I have Oryx robot in our lab. I got my initial crystallization hit from sitting drop vapor diffusion method. This hit can be reproduced by sitting drop through spontaneous nucleation with many tiny crystals after around 1 week but seeding into sitting drop failed and I found once I opened slide and let the drop exposed to air even for a few second, it will turn turbid instantly and I think this might be the reason why seeding in sitting drop is not workable as most protein precipitated and protein concentration dropped very low and seeds implanted inside might be dissolved quickly even after certain periods of equilibration before seeding. That is the reason I want to seperate my drop from air by adding oil on it and then try seeding under oil. Also microbatch under oil might prevent skin formation and maintain protein concentraiton at relatively high level to support seed crystal growth. Best regards, Donghui On Wed, May 19, 2010 at 7:37 PM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: Dear Donghui I can confirm that microseeding with microbatch-under-oil works fine. It’s one of our standard set of scripts. Our Oryx robots use contact dispensing (the tip touches the plate) so the seeds can be added very reliably as a suspension. Several users have reported that the method works, although I don’t know of anyone who has made a statistical comparison between Sitting Drop and Microbatch. I would expect them to be about the same. Do you have an Oryx or some other robot? And was there any particular reason why you want to use microbatch? Best wishes Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, 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 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *wu donghui *Sent:* 19 May 2010 10:29 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] About seeding in microbatch under oil crystallization Hi ccp4bbers, I want to know if anyone has any experience about seeding (streak seeding or microseeding) in microbatch under oil crystallization. I wonder if the oil might block or wipe away the seeds if cat whisker is used for streak seeding. Thank you for your input ahead. Best regards, Donghui
Re: [ccp4bb] About seeding in microbatch under oil crystallization
Hi Donghui, I tried the microseeding using Hampton seeding tool (http://hamptonresearch.com/product_detail.aspx?cid=18sid=157pid=448) for under oil microbatch (without caring about wiping out seed by oil). It is possible to seed the microbatch drop this way. cheers, ravi Ravindra D. Makde Scientific Officer, High Pressure Synchrotron Radiation Physics Division Bhabha Atomic Research Centre, Trombay, Mumbai, India. Tel: +91-22-25506754 (Res.), All that we are is the result of what we have thought. The mind is everything. What we think, we become Buddha --- On Wed, 5/19/10, wu donghui wdh0...@gmail.com wrote: From: wu donghui wdh0...@gmail.com Subject: [ccp4bb] About seeding in microbatch under oil crystallization To: CCP4BB@JISCMAIL.AC.UK Date: Wednesday, May 19, 2010, 5:28 AM Hi ccp4bbers, I want to know if anyone has any experience about seeding (streak seeding or microseeding) in microbatch under oil crystallization. I wonder if the oil might block or wipe away the seeds if cat whisker is used for streak seeding. Thank you for your input ahead. Best regards, Donghui
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Interestingly, Maxwell's demon pops up here, wh... , don't do it. If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed out, you cannot change the #916G of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (#916E double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of GAPDH now make TIM work in one direction. Since many epimerases are very optimized enzymes, why not consider making a fusion with a second enzyme (like a reductase) to make the system flow in one direction. Of course, this depends on what you want to do with the product. Cheers, Michael // /R. Michael Garavito, Ph.D./ /Professor of Biochemistry Molecular Biology/ /513 Biochemistry Bldg. / /Michigan State University / /East Lansing, MI 48824-1319/ /Office:// //(517) 355-9724 Lab: (517) 353-9125/ /FAX: (517) 353-9334Email: rmgarav...@gmail.com mailto:garav...@gmail.com/ // On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a #916G of zero simply corresponded to an equilibrium constant of one. You can certainly have reversible reactions with other equilibrium constants. In fact I think irreversible reactions are simply ones where the equilibrium constant is so far to one side that, in practice, the reaction always goes all the way to product. As Randy pointed out the enzyme cannot change the #916G (or the equilibrium constant). You could drive a reaction out of equilibrium by coupling it to some other reaction which itself is way out of equilibrium (such as ATP hydrolysis in the cell) but I don't think that's a simple mutation of your enzyme. ;-) Dale Tronrud On 05/18/10 00:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the #916G of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/
Re: [ccp4bb] Query - inhibitor screening by fluorescence based assay.
You are measuring fluorescence changes which are likely to be due to compound binding by your enzyme. In this case your blank must be your compound blank and no other. Then you measure changes in fluorescence intensity and lambda max of emission as you add your enzyme. I do not know you system. Note however that binding (1) might be non specific and (2) need not correlate with enzyme inhibition. Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, 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.Mrabetat medecine.uhp-nancy.fr On 19/05/2010 08:48, suresh mattegunta wrote: Dear All, Sorry for the non crystallography related question We are performing a fluorescence based assay to screen for inhibitor compounds of our enzyme and ultimately crystallize the enzyme along with inhibitor. We see that some of our compounds are autofluorescent and thus are effecting fluorescence. In such a case we make a compound blank (assay buffer+ compound) and subtract it from test (enzyme + compound) but still we see the values of test much higher compared to control (only enzyme). In this case can we bring down the values of compound balnk and test by a factor which brings the value of compound blank to the level of blank and compare the values of resultant test value with that of control after subtracting the respective blanks. For example we have the following arbitrary fluorescent readings (AFU) for one compound: Blank (70342) Control (163243) Compound balnk (1865830) Test (2020455) Since compound blank / blank ie (1865830)/(70342) = 26.52 can we do a normalization of compound blank and test values by a factor 26.52, which brings teh value of compound blank to teh level of blank and get the values (1865830)/26.52 = 70355 and 2020455/26.52 = 76186 and say the compound is inhibitor by comparing this test value with the value of control after subtracting teh values of respective blank. Thank you in advance, Suresh -- Suresh V Mattegunta MS Senior Research Associate Discovery Biology Division Aptuit Laurus PVT LTD. ICICI Knowledge Park Hyderabad 500078 India
Re: [ccp4bb] Native Gel Theory and Practice
Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the first direction, then SDS-PAGE in the second one. You actually need both data to infer stoechiometry and subunit composition. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, 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.Mrabetat medecine.uhp-nancy.fr On 19/05/2010 13:01, Jürgen Bosch wrote: Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Native Gel Theory and Practice
Dear Jacob, I know that this is not the answer you were seeking, but for a modest increase in the amount of protein required, a couple of analytical ultracentrifugation experiments would be able to determine stoichiometry and binding affinity for such a system. AUC has the added benefit of being a solution phase system, and you can run it in the buffer of your choice. If your system is as complex as you describe, then native PAGE *might* not unambiguously give you the answers you seek. HTH, David. David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 18 May 2010 21:04, Jacob Keller j-kell...@md.northwestern.edu wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MW Charge MW/Charge A 22 -5 -4308 B 17 -24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Native Gel Theory and Practice
You don't necessarily need the second dimension. BN-PAGE gel: protein A alone| some proteins you know the size as reference|protein B alone| your mixture You will be able to see in the mixture a) one or b) multiple bands, since the Coomassie is equally distributed and attached to your protein in a non-denaturing way it is directly proportional to the molecular mass of your protein complex. I'm searching for the protocol which was I believe either from the Görg group or a group in Berlin, perhaps also Mann demonstrating dissociation of a multimeric complex in dependence of reducing agent. If I find it I will post it tonight. Jürgen On May 19, 2010, at 10:01 AM, Nadir T. Mrabet wrote: Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the first direction, then SDS-PAGE in the second one. You actually need both data to infer stoechiometry and subunit composition. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, 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.Mrabetat medecine.uhp-nancy.fr On 19/05/2010 13:01, Jürgen Bosch wrote: Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Native Gel Theory and Practice
Thanks Jürgen. Yes, you may show dissociation. However, especially if you deal with assembly, then it might be difficult, if not impossible, to tell your exact subunit composition if you runBNP only in the first direction. Jacob mentions the possible occurrence of complex assemblies (AB, BB, ABB, AAB). Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, 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.Mrabetat medecine.uhp-nancy.fr On 19/05/2010 16:05, Jürgen Bosch wrote: You don't necessarily need the second dimension. BN-PAGE gel: protein A alone| some proteins you know the size as reference|protein B alone| your mixture You will be able to see in the mixture a) one or b) multiple bands, since the Coomassie is equally distributed and attached to your protein in a non-denaturing way it is directly proportional to the molecular mass of your protein complex. I'm searching for the protocol which was I believe either from the Görg group or a group in Berlin, perhaps also Mann demonstrating dissociation of a multimeric complex in dependence of reducing agent. If I find it I will post it tonight. Jürgen On May 19, 2010, at 10:01 AM, Nadir T. Mrabet wrote: Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the first direction, then SDS-PAGE in the second one. You actually need both data to infer stoechiometry and subunit composition. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, 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.Mrabetat medecine.uhp-nancy.fr http://medecine.uhp-nancy.fr On 19/05/2010 13:01, Jürgen Bosch wrote: Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu *** - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
[ccp4bb] Postdoctoral opportunity in Structural Biology at Genzyme Corporation (apply online at www.genzyme.com/careers) (requisition ID 21308).
We have a postdoctoral position open in the Fragment-Based Drug Discovery (FBDD) group in Drug and Biomaterial RD at Genzyme Corporation in Waltham, MA. We are seeking a highly motivated recent PhD graduate in Structural Biology for a two-year position with a possible one-year extension. This position offers opportunities to work on structural biology of protein targets of therapeutic importance, involving activities from construct design to structural determination of target protein in complex with substrates or inhibitors. The successful candidate will have access to our cutting edge FBDD technology, including state of the art crystallization, X-ray diffraction system and biophysical analysis platforms. This postdoctoral associate will also have the opportunity to contribute to active discovery programs and gain experience across key aspects of biotech drug discovery as a member of a successful multidisciplinary team. Basic Qualifications: PhD in structural biology or related disciplines (biochemistry, biophysics) with track record of solving X-ray crystal structures of proteins and protein complexes. Experience with construct design, protein purification, protein crystallization, data collection, structure determination and interpretation are essential. Preferred Qualifications: Strong background in biochemistry with in-depth understanding of ligand-protein interactions and familiarity with techniques such as ITC, surface plasmon resonance etc. to characterize proteins in support of structural biology is highly desired. Genzyme Corporation, ranked as one of the foremost biotechnology companies in the world, is committed to providing an exceptional environment in which individuals can excel, and achieve their professional and personal goals. Genzyme Corporation has been selected by FORTUNE magazine as one of the 100 Best Companies to Work For in 2006 in the United States. By applying for a position with Genzyme, you are taking the first step toward becoming a part of our dynamic and talented team, and hopefully sharing in our continued success. If you are interested, please apply online at www.genzyme.com/careers (requisition ID 21308).
Re: [ccp4bb] Native Gel Theory and Practice
Dear Jacob, somewhat adding to list of 'not really answering your question', here is the reference for native page that still uses a dye thereby trying to limit the influence of the charge on the speed. Might be helpful as they discuss some applications. Otherwise AUC really sounds like the way to go. Nat Protoc. 2006;1(1):418-28. Blue native PAGE. Wittig I, Braun HP, Schägger H. best, hannes uchtenhagen Hannes Uchtenhagen Karolinska Institutet Center for Infectious Medicine (CIM) Karolinska University Hospital Huddinge, F59 SE-141 86 Stockholm, Sweden Office: +46-(0)8-524 86981 Mobile: +46-(0)7-36901461 On 2010-05-18 22:04, Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MW Charge MW/Charge A 22 -5 -4308 B 17 -24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] different compilers for ccp4 code
I believe that Francois is correct in saying that a feature of intel compilers is that they do not work well on non-intel CPUs. Can't imagine why that would be. If you have a an intel CPU then using an intel compiler would be an advantage. Code optimization is usually architecture specific, so fiddling with the compiler options may also make a difference. Adam On Tue, 18 May 2010, Francois Berenger wrote: Hello, This is not what you asked for, but I think it is good to know: Intel compilers don't seem to like non-Intel CPUs: http://www.agner.org/optimize/blog/read.php?i=49 Regards, F. Terry Lang wrote: Hey Everyone, I am considering switching from gcc to the Intel compiler in the hopes of making some of calculations run a bit faster. Has anyone ever tried compiling the ccp4 code base with the Intel compilers? Is there a difference in speed? What about in the reproducibility of the calculations? Any changes in statistics? Any information would be greatly appreciated! Thanks, Terry
[ccp4bb] Job Opening for Industrial Post Doc (or other early career) in Xray Crystallography Software Development
Hello All- We have an opening at QuantumBio Inc. for an Industrial Post Doc (or other early career) in Xray Crystallography Software Development. Below is the short job description. Please feel free to contact me if you have any questions or comments about the position. === Contact: Dr. Lance M. Westerhoff, la...@quantumbioinc.com, 814-235-6908 Job Summary: Beginning immediately: QuantumBio Inc., headquartered adjacent to The Pennsylvania State University in scenic State College, PA, is in need of a post doctoral computational or early career Biochemist/Chemist or Xray Crystallographer interested in continuing his or her career within a dynamic, small-company environment. Under the direction of Dr. Lance M. Westerhoff, General Manager of QuantumBio Inc. and with the support of Dr. Kenneth M. Merz Jr., QuantumBio's CSO and Professor at the University of Florida, the successful candidate will work with both academic and industrial partners to help transition and validate the novel, quantum mechanically based Xray refinement technologies recently developed in Dr. Merz's academic laboratory to QuantumBio’s product line. The successful candidate will be integral to this project and work with other members of the team to research and develop the software aspects necessary to validate and deploy this novel technology across the pharmaceutical industry. The position requires the candidate to have a Ph.D. in Chemistry, Biochemistry, or related field and have experience with Phenix, CCP4, or other similar xray refinement packages along with an understanding of modern software development principles. Due to the limited number of new work visas available, only American citizens or candidates with valid work permits will be considered at this time. A willingness to publish is required, and experience with C++ and high performance programming is a plus. Opportunities for advancement may be available through additional SBIR/STTR-style projects, and industry-supported collaborations. Further, in addition to the research and development duties of the project, the position will afford the successful candidate with access to and experience with QuantumBio’s industry partners including a number of pharmaceutical, biotech, and computational firms. Interested candidates should submit at their earliest convenience their CV along with one or two representative publications to the Contact above. About QuantumBio: QuantumBio Inc is accelerating drug discovery efforts by providing pharmaceutical, biotechnology companies and life science research organizations with a next generation of Computer-Assisted Drug Design (CADD) and Computer-Assisted Molecular Modeling (CAMM) solutions based on the increased accuracy and power of quantum mechanics. These solutions include a number of options tailored to each customer’s specific requirements including software licensing, services, and customization. See http://www.quantumbioinc.com/for more! Thank you for your time and interest! -Lance Lance M. Westerhoff, Ph.D. General Manager QuantumBio Inc. WWW:http://www.quantumbioinc.com Email:la...@quantumbioinc.com Phone: 814-235-6908 Fax:814-235-6909
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
You absolutely right, I thought about it. Maia Marius Schmidt wrote: Interestingly, Maxwell's demon pops up here, wh... , don't do it. If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed out, you cannot change the #916G of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (#916E double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of GAPDH now make TIM work in one direction. Since many epimerases are very optimized enzymes, why not consider making a fusion with a second enzyme (like a reductase) to make the system flow in one direction. Of course, this depends on what you want to do with the product. Cheers, Michael // /R. Michael Garavito, Ph.D./ /Professor of Biochemistry Molecular Biology/ /513 Biochemistry Bldg. / /Michigan State University / /East Lansing, MI 48824-1319/ /Office:// //(517) 355-9724 Lab: (517) 353-9125/ /FAX: (517) 353-9334Email: rmgarav...@gmail.com mailto:garav...@gmail.com/ // On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a #916G of zero simply corresponded to an equilibrium constant of one. You can certainly have reversible reactions with other equilibrium constants. In fact I think irreversible reactions are simply ones where the equilibrium constant is so far to one side that, in practice, the reaction always goes all the way to product. As Randy pointed out the enzyme cannot change the #916G (or the equilibrium constant). You could drive a reaction out of equilibrium by coupling it to some other reaction which itself is way out of equilibrium (such as ATP hydrolysis in the cell) but I don't think that's a simple mutation of your enzyme. ;-) Dale Tronrud On 05/18/10 00:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the #916G of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/
Re: [ccp4bb] Native Gel Theory and Practice
That's interesting. Thanks. Maia Nadir T. Mrabet wrote: Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the first direction, then SDS-PAGE in the second one. You actually need both data to infer stoechiometry and subunit composition. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, 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.Mrabetat medecine.uhp-nancy.fr On 19/05/2010 13:01, Jürgen Bosch wrote: Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Help needed in solving a MAD dataset
Hi All, I am new to protein crystallography. I would like to know the steps involved in solving a MAD dataset by using the program in CCP4 where you determine the phases and then obtain the trace. The dataset is collected at 3 different wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous signal. Thanks, Qing Lu
[ccp4bb] Help needed in solving a MAD dataset
Hi All, I am new to protein crystallography. I would like to know the steps involved in solving a MAD dataset by using the program in CCP4 where you determine the phases and then obtain the trace. The dataset is collected at 3 different wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous signal. Thanks, Qing Lu
Re: [ccp4bb] Help needed in solving a MAD dataset
CCP4 way: locate the Se sites with SHELX (if you use the CCP4I gui it's technically a ccp4 program :-) ) Try using only your peak data set first. If you can't locate your sites with the single wavelength then add remote (DAD) and if that doesn't work go MAD. non-ccp4 way run hkl2map as frontend to SHELX, do the same thing. After 10 minutes go and open a bottle of your favourite sparkling cider* and start tracing the structure. Jürgen * can be replaced by other sparkling product containing trace amounts of EtOH supplemented with flavours On May 19, 2010, at 12:01 PM, Qing Lu wrote: Hi All, I am new to protein crystallography. I would like to know the steps involved in solving a MAD dataset by using the program in CCP4 where you determine the phases and then obtain the trace. The dataset is collected at 3 different wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous signal. Thanks, Qing Lu - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Help needed in solving a MAD dataset
First, you will need CCP4 installed and set up properly. You will also need to know your protein sequence. Put the latter into a text file (FASTA format will do). Next, download the file Elves from: http://ucxray.berkeley.edu/~jamesh/elves/download.html then type: chmod a+x Elves Elves /path/to/data/frames/ ./proteinsequence.txt If you don't know how to answer any of the questions Elves ask you, then the default is usually the best choice. If the structure can be solved from the data, this will do the trick most of the time (~80% in my tests). Things they don't handle well are twinning and severe radiation damage. -James Holton MAD Scientist Qing Lu wrote: Hi All, I am new to protein crystallography. I would like to know the steps involved in solving a MAD dataset by using the program in CCP4 where you determine the phases and then obtain the trace. The dataset is collected at 3 different wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous signal. Thanks, Qing Lu
[ccp4bb] translational NCS
Dear CCP4bbs, I am dealing with a case involving pseudo-translational symmetry. I wanted to know what was the simplest way to draw NCS copies of a molecule deduced from the positions I observed in native Patterson. Is there a translate option where on can give fractional coordinates in Coot or Pymol? Thanks for your help! Nicolas
[ccp4bb] Question: Refmac5 stats reported in pdb REMARK 3
Compare these two lines from phenix.refine: REMARK 3 NUMBER OF REFLECTIONS : 46001 REMARK 3 FREE R VALUE TEST SET COUNT : 2339 with those from refmac, ostensibly using the same data and start pdb: REMARK 3 NUMBER OF REFLECTIONS : 43672 REMARK 3 FREE R VALUE TEST SET COUNT : 2339 I know there are 46011 reflections with |F|0 in the files I used. phenix.refine removes 10 of these as outliers. The 46001 remaining reported in REMARK 3 *include* the test set. With REFMAC, 43672+2339=46011 so it appears that Refmac reports just the *working* set count in that first line, excluding the test set. Is this is a bug with one program or the other, or a bug in the PDB definition of REMARK 3 ? http://www.wwpdb.org/documentation/format23/remark3.html This appears to be a source of inconsistency. phenix.refine 1.6-289 refmac5 5.4.0077 (I'm apparently a Luddite) Phil Jeffrey Princeton
Re: [ccp4bb] Question: Refmac5 stats reported in pdb REMARK 3
Phil, I think the PDB documentation in this area (such as it is) is unclear, and it's not easy to fathom what was the original intent. The first line you refer to: REMARK 3 NUMBER OF REFLECTIONS : appears in the section with the sub-heading: REMARK 3 DATA USED IN REFINEMENT. Arguably, and this was my initial impression, the test set is not 'used' in refinement, i.e. it has no effect on the refined parameters (except possibly indirectly via adjustment of the weights). So on that basis, Refmac is correct, the number of reflections 'used' should *exclude* the test set. However the second line you refer to: REMARK 3 FREE R VALUE TEST SET COUNT : appears in the next section with the other cross-validation info, with the sub-heading: REMARK 3 FIT TO DATA USED IN REFINEMENT. and seems to contradict this, since its use of the word 'used' (i.e. in the wider sense of the reflections merely being read in by the refinement program and passing the various rejection tests) clearly does imply that the test set is *included* in the count of 'used' reflections. So it all comes down to what is meant by 'used'. Cheers -- Ian On Wed, May 19, 2010 at 9:02 PM, Phil Jeffrey pjeff...@princeton.edu wrote: Compare these two lines from phenix.refine: REMARK 3 NUMBER OF REFLECTIONS : 46001 REMARK 3 FREE R VALUE TEST SET COUNT : 2339 with those from refmac, ostensibly using the same data and start pdb: REMARK 3 NUMBER OF REFLECTIONS : 43672 REMARK 3 FREE R VALUE TEST SET COUNT : 2339 I know there are 46011 reflections with |F|0 in the files I used. phenix.refine removes 10 of these as outliers. The 46001 remaining reported in REMARK 3 *include* the test set. With REFMAC, 43672+2339=46011 so it appears that Refmac reports just the *working* set count in that first line, excluding the test set. Is this is a bug with one program or the other, or a bug in the PDB definition of REMARK 3 ? http://www.wwpdb.org/documentation/format23/remark3.html This appears to be a source of inconsistency. phenix.refine 1.6-289 refmac5 5.4.0077 (I'm apparently a Luddite) Phil Jeffrey Princeton
[ccp4bb] Postdoctoral Position Available in Structural Biophysics and Protein Engineering in Rockville, Maryland, USA
Postdoctoral Position Available in Structural Biophysics and Protein Engineering in Rockville, Maryland, USA A NIH funded postdoctoral position is available for a highly motivated individual at the Center for the Advanced Research in Biotechnology (CARB) at the University of Maryland Biotechnology Institute in Rockville, Maryland, USA. Several research projects are available to study the interleukin-7 signaling pathway at the structural and biophysical levels. Candidates must have a Ph.D. degree in biochemistry or a related field, with experience in molecular biology, bacterial and insect cell protein expression and purification. Prior experience in structural (X-ray crystallography or NMR spectroscopy) and biophysical techniques (circular dichroism, calorimetry, fluorescence, etc.) is preferred. Salary will be commensurate upon experience. For further information or to apply for the positions please send a cover letter, CV, and two references to Dr. Scott Walsh (email: wal...@umbi.umd.edu).
Re: [ccp4bb] Native Gel Theory and Practice
How about using static light scattering to determine the actual molecular weight or dynamic light scattering to measure the diameter of the complex. Sheemei From: Jürgen Bosch jubo...@jhsph.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 19 May 2010 11:00:24 Subject: Re: [ccp4bb] Native Gel Theory and Practice Here's just one example, which I quickly found from Reisinger and Eichacker. Isolation of membrane protein complexes by blue native electrophoresis. Methods Mol Biol (2008) vol. 424 pp. 423-31 Now Jacob has A 22 kDa B 17 kDa, the charge can be disregarded in BN PAGE. If we do the math for all the theoretical complexes and assume globular shape for all of them.78 AABB (22+22+17+17) 61 AAB (22+22+17) 56 ABB (22+17+17) 39 AB (22+17) 22 A 17 B I'd use a higher percentage gel 10-20% then you should be able to separate the 6 species mentioned above. Jürgen P.S. just trying to be helpful On May 19, 2010, at 11:40 AM, Maia Cherney wrote: Yes, you can separate by electrophoresis, that's why we use it, but we cannot calculate accurate mass of complexes. Maia Jürgen Bosch wrote: Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
