Re: [ccp4bb] Off topic: vector map editing and DNA sequence alignment software
Hello folks, A couple of years ago, we had a related discussion which I cut paste below. I still use Vector NTI which is expensive and bloated, but works well. I've also had good experiences with Gentle which is free and open source. Darren Thanks for the 38 replies, both on and off the bboard. I have tested some of them and my favorites so far are ApE and Gentle which are free and quite good. But there may be others that are also good and I missed. Summary: *Firstly*, good advice from Warren DeLano: 1. Be wary of relying upon free tools not based on open-source code. 2. Be extremely wary of free tools which come with a license manager. 3. Instead favor free software tools which strictly meet the established definitions of: Open Source: http://www.opensource.org/docs/osd, Free Software: http://www.gnu.org/philosophy/free-sw.html, or Public Domain: http://en.wikipedia.org/wiki/Public_domain since it is *only* those tools that can be safely taken for granted over the long haul. But be prepared to pay good money for good software! *Secondly*, if you are going to stop using VectorNTI, export valuable files in .gb format before the program locks. If this happens, contact Invitrogen and they (might) issue a short time extension as they did for me. *Recommended programs:* *Geneious *and *CLCbio *workbench are professional polished products competing with VectorNTI – but CLC free version is just a plasmid viewer really. Sebastiano and others - much much easier than VectorNTI is *ApE *( http://www.biology.utah.edu/jorgensen/wayned/ape/), which is multi-platform and very easy to use for simple tasks. I tried ApE and was really impressed, once I got past the very simple looking format. This would do most of the things required for designing vectors and works with .gb format files – Darren *Serial cloner *(http://serialbasics.free.fr/Serial_Cloner.html) suggested by James Stroud. It works only with fasta or .xdna files – so is really a DNA editor and seems to have limited Protein analysis functions, even displaying translated ORFs above DNA sequence. But splicing DNA sequences together seems efficient. Mark Brooks - recommended *BioEdit*: http://www.mbio.ncsu.edu/BioEdit/bioedit.html It has an old fashioned cluttered interface, but does do sequence editing, translation into proteins, ClustalW alignments and contig assemblies (a bit like ContigExpress in Vector NTI). It opens ABI files for sequencing data, to view the chromatograms. It uses the external programs such as clustalw alignments or cap3 to do the contig assemblies, and its licence doesn't expire! For storing everything, I put my primers, plasmid sequences, insert sequences in a MySQL database, with an HTML front end I wrote: http://plasmidb.sourceforge.net/ *Plasmi::db *also has a homespun feel to it, and only works with Firefox, for example (not other browsers). There is a primer designer page, for traditional cloning by restriction digestion etc.. I can't pretend it's in the same league as Vector NTI, though. The data is stored in a non-proprietary format; database tables which can be viewed with either the HTML pages, or MS Excel, for example. Andy Gulick recommends the *Workbench* suite at the *San Diego Supercomputer Center*. It allows you to maintain a database of protein and DNA sequence, has many tools, and allows you to create subprojects to help organize. http://workbench.sdsc.edu Yong-Fu Li suggested *Lasergene*, but not enthusiastically due to requirement to reformat files and not very good editing functions. Roger Dodd - *PlasmaDNA *which seems pretty good for the basics http://research.med.helsinki.fi/plasmadna/ . Christian Biertümpfel recommends another free tool: *pDRAW32 *( http://www.acaclone.com/ ). It runs natively under Windows and with the emulator wine on Linux. Francis Reyes - Not sure if it's been mentioned, but I personally use *EnzymeX *(http://mekentosj.com/enzymex/) .Also recommends PDF library organizer Papers (http://mekentosj.com/papers/) to be exceptional. Juan Sanchez Weatherby - GCK2 (*GeneConstructionKit*) and another * GeneInspector*. They where pretty amazing and with lots of features for plasmid design, keeping history, sharing, and lots more. I suppose they must have improved quite a lot over the years. I can't remember what the license was like (money wise) but I think you can download a free version (doesn't let you save or print things but shows what you can actually do with them). The link you need to find them is http://www.textco.com/products/index.html Bryan Lepore – Lots can be done just with with [1] *expasy tools *and [2] *sequence manipulation suite*, which is entirely downloadable for local use. http://www.bioinformatics.org/sms2/about.html (Darren says: I agree *SMS *is very useful indeed and can be run via their website – no installation) There is *GENtle *which has a whole slew of tools associated with it. There are versions for several platforms.
Re: [ccp4bb] Fwd: P21/n spacegroup
You wrote: Original Message Subject: P21/n spacegroup Date: Tue, 27 Sep 2011 14:38:28 +0100 From: Franck franck.co...@cnrs-orleans.fr Dear all, i'm struggling trying to integrate x-ray data in spacegroup P 1 21/n 1 (racemic mixture of peptides) with XDS or Imosflm. Can someone tell me the best way to do it ? Thanks in advance. Sincerely, Franck. Franck, struggling is rather unspecific. Is there an error message from XDS, or anything else that tells you that something's wrong? The lists in IDXREF.LP and CORRECT.LP that start with POSSIBLE SPACE-GROUPS FOR PROTEIN CRYSTALS do not list _all_ spacegroups, just the protein ones. But XDS handles all 230 spacegroups. Just run INIT COLSPOT IDXREF in spacegroup 0 (which means unknown) and inspect the tables. Then input the correct spacegroup number and the mP cell parameters as found. Run IDXREF DEFPIX INTEGRATE CORRECT. HTH, Kay -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature smime.p7s. smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] Apparent twinning in P 1 21 1
You might like to look at this.. It tries to explain likely twinning possibilities in P21. If you get C and P21, then probably a~=c - then Beta can have any value. C222 axes are then always possible with a* +c* , a*-c*, b* all having angles ~ 90 Without twinning you wont get 222 symmetry though. Pointless helps here. Eleanor http://www.ccp4.ac.uk/dist/html/twinning.html On 09/27/2011 08:23 PM, Linda Schuldt wrote: Dear Yuri, in a monoclinic space group an orthorhombic lattice metric can be simulated when one of the following conditions is fulfilled: i) a = c [e.g. in Wittmann Rudolph (2007) Acta Cryst. D63, 744-749], ii) the beta angle is close to 90° [e.g. in Larsen et al. (2002) Acta Cryst. D58, 2055-2059 ] or iii) c cos beta is about -a/2 [e.g. in Declercq Evrard, (2002) Acta Cryst. D57, 1829-1835]. The a and b axes of the orthorhombic cell are identical to the monoclinic a and c axes, respectively. The length of the orthorhombic b-axis can also be calculated by c(monoclinic) cos(beta-90°) = 1/2b(orthorhomic). I would assume that you have the case iii with a quite high twin fraction. If I recall correctly, Declercq and Evrard have a nice figure in their paper showing the geometric relationship. If not, let me know and I can sent you a figure. Good luck! Linda Yuri Pompeu schrieb: Hello everyone, I have a 2.3A data set that could be scaled in C 2 2 21 and P 1 21 1 Intensity statistics tests indicate twinning (pseudo-merohedral h,-k,-h-l in P 1 21 1) I find a good MR solution and when I try to refine it with the twin law I get fairly good maps and decent Rs 21-28%. I can see features tha were not in the search model Which leads me to think that this a valid solution. The one thing that bothers me however is the fact that my beta angle in P 1 21 1 is 104 (not close to 90) and that the geometry gets worse after refinement? Any suggestions? cheers *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark
[ccp4bb] PhD studentship in Terahertz Spectroscopy
We are looking for an outstanding PhD student to apply Terahertz Spectroscopy to the study of protein librations and folding. The studentship is funded at the standard research council rate for 3 years. A first class or upper second class degree (or equivalent) is essential. For further details contact Robert Donnan robert.don...@eecs.qmul.ac.uk or myself. To apply, please email your cv to r.w.pickersg...@qmul.ac.uk (please put ?terahertz PhD? on the subject line); applications will be accepted until the 15th October and we should like to start this studentship January 2012 or earlier. Richard W Pickersgill Professor of Structural Biology School of Biological and Chemical Sciences Queen Mary University of London London E1 4NS England Telephone 02078828444
Re: [ccp4bb] Protein melting temperatures
I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Postdoctoral position
We seek to recruit an outstanding postdoctoral scientist with strong interest in Structural Biology/Structural Enzymology. The main goal of this project is to illuminate the chemistry of cobalamin biosynthesis in molecular and mechanistic detail exploiting our recent discovery that several of the biosynthetic enzymes trap their products. We want to understand how intermediates in the biosynthetic pathway are passed from one enzyme to the next. We shall do this using a combination of structural, biochemical, biophysical and genetic approaches. This is a four-year, full time position, funded by the BBSRC, starting in November 2011 or as soon as possible thereafter. The salary is in the range of £30,350 - £33,794 per annum and is inclusive of London allowance. Benefits include 30 days annual leave, defined benefit pension scheme and an interest?free season ticket loan. The successful candidates will hold a PhD in Structural Biology/Biochemistry or have equivalent experience and will have experience in producing and purifying proteins. A significant publication record would be an advantage. Candidates must be able to demonstrate their eligibility to work in the UK in accordance with the Immigration, Asylum and Nationality Act 2006. Where required this may include entry clearance or continued leave to remain under the Points Based Immigration Scheme. Informal enquiries can be made to Professor Richard W Pickersgill (r.w.pickersg...@qmul.ac.uk or 020-7882-8444). Further details and an application form can be obtained from the Human Resources website on: http://www.hr.qmul.ac.uk/vacancies. For further information about the School, please see http://www.sbcs.qmul.ac.uk. Complete application forms must not be sent directly to Prof Pickersgill but should be returned, together with a copy of your CV, to Ms Sunita Devi-Paul, School of Biological Chemical Sciences, Queen Mary, University of London, Mile End Road, London, E1 4NS, or by e-mail: sbcs-vacanc...@qmul.ac.uk. Please quote reference number 11226/NL. The closing date for applications is 13 October 2011 at 4.00pm. Richard W Pickersgill Professor of Structural Biology School of Biological and Chemical Sciences Queen Mary University of London London E1 4NS England Telephone 02078828444
Re: [ccp4bb] Protein melting temperatures
For what it's worth, we've been using thermofluor to compare the 'apparent' melting points of enzymes with their thermal stability measured as inhibition of their respective reactions by elevated temperature. The data so far make sense - the differences in apparent enzyme Tm (using the same conditions as the reaction mix!) match the differences in the half-inhibition T. Not the absolute number,though (which is not unexpected givn the different kinds of measurements involved). So I'd say thermofluor is reasonably good at comparing different proteins. Qualitatively at least. Artem On Wed, Sep 28, 2011 at 6:25 AM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Protein melting temperatures
We have proteins that melt at 60˚C but they don't crystallize. According to your 45 degree rule we should have crystals, what are we doing wrong ? Jürgen On Sep 28, 2011, at 10:05 AM, Artem Evdokimov wrote: For what it's worth, we've been using thermofluor to compare the 'apparent' melting points of enzymes with their thermal stability measured as inhibition of their respective reactions by elevated temperature. The data so far make sense - the differences in apparent enzyme Tm (using the same conditions as the reaction mix!) match the differences in the half-inhibition T. Not the absolute number,though (which is not unexpected givn the different kinds of measurements involved). So I'd say thermofluor is reasonably good at comparing different proteins. Qualitatively at least. Artem On Wed, Sep 28, 2011 at 6:25 AM, Patrick Shaw Stewart patr...@douglas.co.ukmailto:patr...@douglas.co.uk wrote: I actually think you can make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlmailto:a.perra...@nki.nl wrote: Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukmailto:patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.ukhttp://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034tel:1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Rigaku high voltage tank
On Sep 28, 2011, at 7:15 AM, Artem Evdokimov wrote: Are you indeed referring to the oil-filled high voltage transformer core in the older model generators? Yes. (Newer ones tend to use solid state voltage multipliers). That sounds much more sane. I recall that there were issues with those things that were eventually traced to elevated humidity. Now, that was humidity around 80% which should not be the case for most systems these days since most of us seem to prefer humidity controlled environments (for the sake of the cryostreams) but it might be worth checking some local leaks etc. Well, I'm at DIY-U, so this very likely is a potential source of the problem. We have to supply our own hamsters for the electrical generators, too. One more reason that the tank transformer might go bad would be inadequate matching between power input and drain, especially high-drain situation caused by an undetected high current (like a parasite current too weak to cause arcing, but strong enough to damage transformer over time). Did your/Rigaku's engineer check the voltages and currents in the tube tower? Do you have the option to record currents while no one is attending the instrument (in the even that the problem is intermittent)? I don't think anything like that was ever done, but I am not sure. We shipped it back to them, and they need more money to continue diagnosis, so we are probably just going to cut our losses on this decade-long nightmare. Artem On Tue, Sep 27, 2011 at 8:51 AM, Patrick Loll pat.l...@drexel.edu wrote: Bill, What do you mean by high voltage tank? When I hear this term, I think of the oil- (and PCB-) filled tank housing the transformer on an old generator; but there's nothing like that on the R-Axis. Do you mean the blue box housing the detector controller? If so, then I can tell you that we've had ours plugged into a 1000 VA UPS (APC Back UPS Pro) for 10 years, with never a glitch (and I'm told that Philly power is not pretty). If instead you're referring to the high voltage tank on the generator, then I have no idea of what to do (although replacing the power cable doesn't sound like a bad idea)...you'd probably need to steal an entire 7-11 to pay for a UPS large enough to condition power for that. Pat On 26 Sep 2011, at 10:15 PM, William G. Scott wrote: Hi Citizens: We seem to run through high voltage tanks on our Raxis IV like guano goes through a goose. Has anyone else had this problem, and, more importantly, what is the best way to protect them. I am assuming it might have something to do with our electrical supply, which is a bit unreliable. Also, does anyone have an extra used functional one they want to get rid of? We've run out of 7-11s to rob to pay for this, and our friendly and helpful radiation safety staff think the best way to deal with this problem is to hack apart the power cable, so it is a current (so to speak) source of frustration. Thanks in advance. Bill William G. Scott Professor Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 228 Sinsheimer Laboratories University of California at Santa Cruz Santa Cruz, California 95064 USA William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/
Re: [ccp4bb] Protein melting temperatures
Dear Raji, what exactly do you mean when you say the melting temperature is 45deg. Did you only test one buffer, or did you test many buffers and 45deg is the most stable one? If you have only tested one buffer you should run a screen testing different buffer systems (pH) and e.g. NaCl concentration and glycerol concentrations (or ligands, if your proteins binds any). Then you identify the buffer which is stabilizing your protein the most. I have seen big impacts on protein stability and crystallization when optimizing my buffers like this. I think you should not only consider the melting temperature alone, but also how the curve looks like. Do you get a high initial flourescence (which often indicates partially unfolded protein or hydrophobic patches) or do you have very low initial flourescence (which is a good sign for compact protein). Another thing to look at is if your transition is sharp (the steeper the better). Taking all this together you can judge if your protein is happy or not. Hope this helps you! Linda Patrick Shaw Stewart wrote: I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark
Re: [ccp4bb] Protein melting temperatures
This paper on thermofluor is a good reference and if you have access to a real time PCR machine, different buffer systems, like the PACT screen can be evaluated within an hour to find out the buffer in which your protein is most stable. It gives the Tm of your protein and if you have a high fluorescence to start with, it means your protein is unfolded to start with. Curr Protoc Mol Biol. http://www.ncbi.nlm.nih.gov/pubmed/21472694# 2011 Apr;Chapter 10:Unit10.28. The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Phillips Khttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Phillips%20K%22%5BAuthor%5D , de la Peña AHhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22de%20la%20Pe%C3%B1a%20AH%22%5BAuthor%5D . Best regards Shankar On Wed, Sep 28, 2011 at 11:03 AM, Linda Schuldt lschu...@mb.au.dk wrote: Dear Raji, what exactly do you mean when you say the melting temperature is 45deg. Did you only test one buffer, or did you test many buffers and 45deg is the most stable one? If you have only tested one buffer you should run a screen testing different buffer systems (pH) and e.g. NaCl concentration and glycerol concentrations (or ligands, if your proteins binds any). Then you identify the buffer which is stabilizing your protein the most. I have seen big impacts on protein stability and crystallization when optimizing my buffers like this. I think you should not only consider the melting temperature alone, but also how the curve looks like. Do you get a high initial flourescence (which often indicates partially unfolded protein or hydrophobic patches) or do you have very low initial flourescence (which is a good sign for compact protein). Another thing to look at is if your transition is sharp (the steeper the better). Taking all this together you can judge if your protein is happy or not. Hope this helps you! Linda Patrick Shaw Stewart wrote: I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark
Re: [ccp4bb] Protein melting temperatures
Susan and everyone, I should apologise for any confusion that I may have caused. Rajiv actually asked his question a year ago, and I accidentally replied to it a year too late! It's an interesting question though Patrick On Wed, Sep 28, 2011 at 5:03 PM, Linda Schuldt lschu...@mb.au.dk wrote: ** Dear Raji, what exactly do you mean when you say the melting temperature is 45deg. Did you only test one buffer, or did you test many buffers and 45deg is the most stable one? If you have only tested one buffer you should run a screen testing different buffer systems (pH) and e.g. NaCl concentration and glycerol concentrations (or ligands, if your proteins binds any). Then you identify the buffer which is stabilizing your protein the most. I have seen big impacts on protein stability and crystallization when optimizing my buffers like this. I think you should not only consider the melting temperature alone, but also how the curve looks like. Do you get a high initial flourescence (which often indicates partially unfolded protein or hydrophobic patches) or do you have very low initial flourescence (which is a good sign for compact protein). Another thing to look at is if your transition is sharp (the steeper the better). Taking all this together you can judge if your protein is happy or not. Hope this helps you! Linda Patrick Shaw Stewart wrote: I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Formulatrix Job Openings: European Sales Engineer, European Support Engineer.
Hello CCP4, Formulatrix has two job openings in Europe. A support engineer and a sales engineer. Candidates may be located anywhere in Europe to be considered for these positions. For more detailed information, including how to apply, click here http://www.formulatrix.com/careers/careers-eu.html. -Jeremy Jeremy Stevenson President, Formulatrix, Inc. www.formulatrix.com jer...@formulatrix.com Mobile +1-781-258-8105
[ccp4bb] Postdoctoral Fellowship in Membrane Protein Structural Biology at NIH
Structural studies on glutamate receptor ion channels: A postdoctoral position in X-ray crystallography is available immediately in the group of Dr. Mark L. Mayer, in the Laboratory of Cellular and Molecular Neurophysiology at the NIH in Bethesda MD, USA. This opening is part of an established program with a long term focus on glutamate receptor ion channels: see http://snb.nichd.nih.gov/index.htm for recent publications and research interests. Substantial efforts have already been undertaken in construct design, protein expression and detergent screening, leading to very promising leads for crystallization. The laboratory is located in the main NIH campus. We have shared state-of-the-art facilities large scale eukaryotic cell culture, and for crystallization, in-house X-ray data collection and other biophysical approaches. We also have regular synchrotron beamtime at the Advanced Photon Source (APS) through membership of SER-CAT. Interested applicants must have a Ph.D. in biochemistry, molecular biology or structural biology, extensive experience in protein expression and purification, and X-ray crystallography. Experience working with insect cell culture and baculovirus expression systems would be an advantage is but not essential. The ideal candidate will be highly motivated, possess excellent communication skills and the ability to work in a collaborative and team-oriented environment. To apply, please e-mail a CV including a list of publications, a brief statement of experience and scientific interests, and contact information for three references to may...@mail.nih.gov Mark Mayer Ph.D. LCMN NICHD NIH DHHS Bldg 35, Room 3B 1002 MSC 3712 35 Lincoln Drive Bethesda MD 20892 3712 --
[ccp4bb] Linux vs MacOS for crystallographic software
Dear colleagues, I need some advice for a new computer. (1) I have the option of an HP Z210 8 GB with a low end Quadro Nvidia 400 512 MB. --How does Coot run with this card? --I am happy with any Linux. However, the system needs updates for security purposes (the University requires it). Do I have to remake the NVidia driver every time there is a kernel update or is there a way around it for this NVidia card? Do you suggest another NVidia card (inexpensive) that is good for coot and automatically updates when the kernel is updated? (2) Second option is an IMAC 4 GB 2.5 GHz with AMD Radeon HD 6750M 512 MB GDDRS. --How does Coot work with this graphics card? --Should I get more memory for Lion? --Is this platform advisable for crystallographic software for the next four years? Thank you in advance for any advice. Jackie Vitali Cleveland State University
Re: [ccp4bb] Linux vs MacOS for crystallographic software
On Wed, Sep 28, 2011 at 5:26 PM, Jacqueline Vitali jackie.vit...@gmail.comwrote: (2) Second option is an IMAC 4 GB 2.5 GHz with AMD Radeon HD 6750M 512 MB GDDRS. --How does Coot work with this graphics card? I don't have exactly this, but something very similar (6770M), and it works very well for me so far, but I'm not a power user. And I definitely don't use stereo, which I assume the iMac won't do. --Should I get more memory for Lion? Yes, absolutely - double it for sure. (Don't upgrade when ordering from Apple unless you have money to burn - you can buy the memory elsewhere for a fraction of the price.) Most of the time you will not need 8GB, but when you want to use all four processor cores at once for refinement etc., it is essential. --Is this platform advisable for crystallographic software for the next four years? For a workstation (or laptop), yes (for clusters/servers I would still recommend Linux). I don't know of any macromolecular crystallography programs that don't run on Mac - my only real complaint is that I really preferred running Coot on Linux (Apple's fault). However, if you care about having hardware stereo, there may be other issues - I have no experience with this. -Nat
Re: [ccp4bb] Linux vs MacOS for crystallographic software
On Sep 28, 2011, at 5:26 PM, Jacqueline Vitali wrote: Dear colleagues, I need some advice for a new computer. (1) I have the option of an HP Z210 8 GB with a low end Quadro Nvidia 400 512 MB. --How does Coot run with this card? OK --I am happy with any Linux. However, the system needs updates for security purposes (the University requires it). Do I have to remake the NVidia driver every time there is a kernel update or is there a way around it for this NVidia card? Do you suggest another NVidia card (inexpensive) that is good for coot and automatically updates when the kernel is updated? Try Ubuntu (or Kubuntu or Xubuntu, etc). You can have Linux and proprietary drivers. (2) Second option is an IMAC 4 GB 2.5 GHz with AMD Radeon HD 6750M 512 MB GDDRS. --How does Coot work with this graphics card? OK --Should I get more memory for Lion? Max it out. Lion is a memory pig. --Is this platform advisable for crystallographic software for the next four years? It is essentially BSD unix. So it should be fine, unless they continue to lobotomize everything and make it into an iPod on a stick. Thank you in advance for any advice. Jackie Vitali Cleveland State University
Re: [ccp4bb] Linux vs MacOS for crystallographic software
I use home built LINUX Intel boxes with 2Gb memory and low end Nvidia cards (GT 8000 series to GT 200 series) and they are fine with Coot, Pymol, CCP4i. I can even run CrysalisPro (Windows) in WINE. More memory is cheap to add. If you use a Ubuntu LTS release, you get 3 yr of updates. Proprietary Nvidia binaries can be downloaded from the Ubuntu repos, and they will reinstall every time you do a kernel update. Ubuntu has been pretty painless to maintain compared to Fedora, at least when I made the switch. Roger Rowlett On Sep 28, 2011 8:27 PM, Jacqueline Vitali jackie.vit...@gmail.com wrote: Dear colleagues, I need some advice for a new computer. (1) I have the option of an HP Z210 8 GB with a low end Quadro Nvidia 400 512 MB. --How does Coot run with this card? --I am happy with any Linux. However, the system needs updates for security purposes (the University requires it). Do I have to remake the NVidia driver every time there is a kernel update or is there a way around it for this NVidia card? Do you suggest another NVidia card (inexpensive) that is good for coot and automatically updates when the kernel is updated? (2) Second option is an IMAC 4 GB 2.5 GHz with AMD Radeon HD 6750M 512 MB GDDRS. --How does Coot work with this graphics card? --Should I get more memory for Lion? --Is this platform advisable for crystallographic software for the next four years? Thank you in advance for any advice. Jackie Vitali Cleveland State University
Re: [ccp4bb] Linux vs MacOS for crystallographic software
On 09/29/2011 09:46 AM, William G. Scott wrote: On Sep 28, 2011, at 5:26 PM, Jacqueline Vitali wrote: Dear colleagues, I need some advice for a new computer. (1) I have the option of an HP Z210 8 GB with a low end Quadro Nvidia 400 512 MB. --How does Coot run with this card? OK --I am happy with any Linux. However, the system needs updates for security purposes (the University requires it). Do I have to remake the NVidia driver every time there is a kernel update or is there a way around it for this NVidia card? Do you suggest another NVidia card (inexpensive) that is good for coot and automatically updates when the kernel is updated? Try Ubuntu (or Kubuntu or Xubuntu, etc). You can have Linux and proprietary drivers. By the way, how about some Debian packages for CCP4? That would make it installable painlessly on both Ubuntu and Debian systems for academic users, which would be quite cool. Regards, F.
Re: [ccp4bb] Off topic: vector map editing and DNA sequence alignment software
Dear CCP4Pers, also j5 might be a good choice. I am not personally familiar with the software, so I would happy to hear any comments. It also seems to cope with modern cloning techniques like SLIC. clicking away at http://j5.jbei.org/index.php/Main_Page Regards, Juha On 27 September 2011 18:42, Florian Schmitzberger schmitzber...@crystal.harvard.edu wrote: Dear All, What type of software are people commonly using these days for vector/plasmid map editing, making/visualizing vector maps, and aligning (small to medium size) DNA sequencing data? Preferably, it should not be too expensive and be able to write text files, readable by other programs. I am familiar with VectorNTI, which is great for vector visualization and editing; but I find it somewhat expensive. Sequencher seems good to quickly align DNA sequences (such as from DNA sequencing) with templates, but is not free. I have been using ApE for while for alignments, but aligning many sequences is more cumbersome than in Sequencher; I have not tested if Sequencher is good at visualizing and editing plasmid maps. Ideally, I would like to have a single program for both purposes (vector editing and DNA sequence comparison). Does something like that exist? What are the alternatives to above programs? Thank you in advance. Florian --- Florian Schmitzberger, PhD Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, Seeley G. Mudd 123 Boston, MA 02115, US Tel: 001 617 432 5603 -- Genius may have its limitations, but stupidity is not thus handicapped. -Elbert Hubbard Juha Vahokoski Kalliotie 16 as. 10 90500 Oulu Finland mobile:+358 40 5286 778
