Re: [ccp4bb] static linking of ccp4 libs
Hi,
these are still shared libraries - just that they don't want to load
any other shared library. Have a look at
% file libccp4c.so libccp4c.a
% ldd libccp4c.so libccp4c.a
to compare static library (*.a) and shared one (*.so). You'll see the
difference. So everything is fine and as expected.
Cheers
Clemens
On Thu, Feb 26, 2009 at 12:26:23PM +0100, Justin Lecher wrote:
> Hi all,
>
> hopefully reaches some devs who can help me.
>
> I try to build shared libs for ccp4 using the configure command
>
> ./configure \
> --onlylibs \
> --with-shared-libs \
> --with-fftw=/usr \
> --with-warnings \
> --disable-cctbx \
> --tmpdir="${TMPDIR}" \
> linux
>
> This should build the libs as shared libs and not statically linked. Am
> I right?
> But what I see is following
>
> $ ldd libccp4c.so
> statically linked
>
> $ ldd libccp4f.so
> statically linked
>
> ...
>
> Am I doing something wrong or is this a build system problem? I attach
> the full build.log
>
> Thanks,
>
> justin
>
>
>
>
> --
> Justin Lecher
> Institute for Neuroscience and Biophysics
> INB 2 - Molecular Biophysics II
> Research centre Juelich GmbH,
> 52425 Juelich,Germany
> phone: +49 2461 61 5385
--
***
* 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)
***
[ccp4bb] ccp4 install script
Hello, trying to compile CCP4 6.1.1 on a Solaris machine, I just came across two scripting errors in install.sh: 1. The syntax used in manifest.sh is valid for bash, not for the boune shell called in install.sh; should use SET and EXPORT commands sequentially. 2. After asking for path names, the script seems to be looking for programs.tar (ERROR: unable to find Programs package!!!), which no longer exists since the packages are present as individual tar.gz files. Regards, Oliver --- Dr. Oliver H. Weiergraeber Institute of Structural Biology and Biophysics Molecular Biophysics Research Centre Juelich D-52425 Juelich Germany Phone: +49-2461-612028 Fax: +49-2461-612020 --- --- --- Forschungszentrum Juelich GmbH 52425 Juelich Sitz der Gesellschaft: Juelich Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498 Vorsitzende des Aufsichtsrats: MinDir'in Baerbel Brumme-Bothe Geschaeftsfuehrung: Prof. Dr. Achim Bachem (Vorsitzender), Dr. Ulrich Krafft (stellv. Vorsitzender), Prof. Dr. Harald Bolt, Dr. Sebastian M. Schmidt --- ---
Re: [ccp4bb] off-topic detergent hydrolysis?
I wouldn't use any stock solution that has been sitting around for a year at 4C. Why take the risk that something has happened with it/grown in it? Use a freshly made solution and do not store it at 4C. Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -Original Message- From: CCP4 bulletin board on behalf of deliang Sent: Wed 2/25/2009 9:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic detergent hydrolysis? Hi there, I am using a stock solution of Octyl-Glucoside to crystallize my membrane protein, which was made ~1 year before and kept at 4c . I wonder whether OG can be hydrolysed under this condition and whether there is a way to test. Many thanks, Deliang
[ccp4bb] another truncate question
hi folks i've almost got my B-factors questions sorted out. one thing i still don't understand is this: in truncate's wilson plot, what is plotted on the vertical axis is not ln I_mean, but ln (I_mean/ff_mean) and ff_mean (or Mn_ff, as truncate calls it) is the "average expected value of ff". my question is how is this value calculated? its resolution dependence obviously affects the B-factor, so i would like to understand at least what goes into it. at this point in the analysis, all truncate knows is the cell/spacegroup, and the approximate number of AAs right? is this just an expectation of how the amplitudes ought to fall off vs spacial frequency, without knowing anything about the detailed structure? thanks!
[ccp4bb] updated XDS binaries available
Dear all, please note that the current XDS package binaries won't work after Mar 31, 2008. Binaries which expire Dec 31, 2009 are now available from the XDS website, http://www.mpimf-heidelberg.mpg.de/~kabsch/xds/ . For a list of changes please check out the Release Notes. best wishes, 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] protein folds
The original poster may be interested in this paper from Phil Bourne's lab a few years ago which looked at the contribution of folds (among other things) by the Protein Structure Initiative, particularly Fig 5 and table 1. http://helix-web.stanford.edu/psb04/bourne.pdf "The Status of Structural Genomics Defined Through the Analysis of Current Targets and Structures" P.E. Bourne, C.K.J. Allerston, W. Krebs, W. Li, I.N. Shindyalov, A. Godzik, I. Friedberg, T. Liu, D. Wild, S. Hwang, Z. Ghahramani, L. Chen, and J. Westbrook cheers, Andy 2009/2/26 : > CCP4 bulletin board wrote on 02/25/2009 03:08:10 > PM: > >> On Wednesday 25 February 2009 08:20:14 Jayashankar wrote: >> > Dear Folks, >> > >> > The last novel proteins fold were from the yr 2007(pdb statistics), >> > From 2007 to till date no novel fold has been identified, >> >> If you reached this conclusion by looking at the PDB web site, >> you should note that the site explains these numbers are taken from SCOP. >> The SCOP website states that the most recent update was some time in > 2007. >> >> So you would have to look elsewhere for new folds deposited since > mid-2007. >> >> > > In fact, you have to look no further than SCOP! Or should I say, pre-SCOP, > the pre-release partial classification of the latest batch of proteins from > the PDB. Here's the webpage: http://www.mrc-lmb.cam.ac.uk/agm/pre-scop/ > > This says that they're still classifying proteins deposited as recently as > Oct 2008, which is a relief to me - I was worried that SCOP had been > abandoned. > > To address the original question, here are some numbers: > > Folds in SCOP 1.71 (18 Jan 2005): 971 > Folds in SCOP 1.73 (26 Sep 2007): 1086 > Folds in pre-SCOP (Oct-ish 2008): 1392 > > We're not done finding new folds yet, folks... > > - Matt > > -- > Matthew Franklin , Ph.D. > Senior Scientist, ImClone Systems > 180 Varick Street, 6th floor > New York, NY 10014 > phone:(917)606-4116 fax:(212)645-2054 > > > > Confidentiality Note: This e-mail, and any attachment to it, contains > privileged and confidential information intended only for the use of the > individual(s) or entity named on the e-mail. If the reader of this e-mail > is not the intended recipient, or the employee or agent responsible for > delivering it to the intended recipient, you are hereby notified that > reading it is strictly prohibited. If you have received this e-mail in > error, please immediately return it to the sender and delete it from your > system. Thank you. >
Re: [ccp4bb] protein folds
Concerning the number of proteins folds in existence vs. the number of folds already identified: Ed Berry had some good points regarding sample statistics, and I assume the mathematics of that sort of thing is formalized somewhere. The number of examples per protein fold will be skewed by the common use of molecular replacement for phasing and the tendency of researchers working in one field to solve several related structures. Keep in mind also the mechanism through which new folds have evolved; i.e. through the generation of new genes via duplication, insertion, deletions, point mutations, and the occasional frame shift. That should be enough text to give the impression that I am a serious person with serious ideas, without conveying any actual useful information; so that I now feel justified in changing the topic to a similar question in another field which was published a few years ago, work done by one of those ubiquitous and prolific Dodsons: Estimating the diversity of dinosaurs Steve C. Wang & Peter Dodson PNAS September 12, 2006 vol. 103 no. 37 13601-13605 Published online before print September 5, 2006, doi: 10.1073/pnas.0606028103 http://www.pnas.org/content/103/37/13601.abstract Cheers, - === You can't possibly be a scientist if you mind people thinking that you're a fool. - Wonko the Sane === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu On Thu, 2009-02-26 at 14:19 +, ANDY DODDS wrote: > The original poster may be interested in this paper from Phil Bourne's > lab a few years ago which looked at the contribution of folds (among > other things) by the Protein Structure Initiative, particularly Fig 5 > and table 1. > > http://helix-web.stanford.edu/psb04/bourne.pdf > > "The Status of Structural Genomics Defined Through the Analysis of > Current Targets and > Structures" > > P.E. Bourne, C.K.J. Allerston, W. Krebs, W. Li, I.N. Shindyalov, A. > Godzik, I. Friedberg, T. > Liu, D. Wild, S. Hwang, Z. Ghahramani, L. Chen, and J. Westbrook ...
[ccp4bb] how to show double conformation in PyMol
Hi all, There is a 10 residues loop in my structure which has two conformations, how could I show the double conformations at the same time in ribbon or cartoon model in PyMol? Thanks! yamei
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
We haven't tried SUMO, but had some frustrating results with GST fusions. They did improve expression and solubility - BUT in one case the target protein precipitated immediately when the tag was cleaved off, and resisted all attempts to bring it back to life. In another case, the fusion protein dragged chaperones into the prep that were nearly impossible to get rid of completely, thus ruining our ATPase assays. Is SUMO, being smaller, less likely to drag such crud along with it? Phoebe Original message >Date: Wed, 25 Feb 2009 14:48:57 -0500 >From: Mo Wong >Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli >To: CCP4BB@JISCMAIL.AC.UK > > Thanks to all who responded. Actually, this bulletin > board is better for help with molecular biology than > the molecular biology bulletin board I am subscribed > to! > > On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks >wrote: > > Mo, > Just to add my 50 cents, I didn't see any > mention of the use of fusion proteins in your > original post. GST, MBP or my personal, and > completely biased, favourite SUMO (plus many more > proteins) have been shown to enhance expression > when fused to the amino terminus of a target > protein. If you fear you have toxicity, simply > tracking the OD600 pre and post induction normally > tell you if this is happening. I've worked with > proteins that basically baselined the cell growth > upon induction and, as Artem stated, at least I > knew my protein was being made albeit at very low > levels. > > Stephen > > -- > Stephen Weeks, Ph. D. > Drexel University College of Medicine > Department of Biochemistry and Molecular Biology > Room 10102 New College Building > 245 N. 15th St. > Philadelphia, PA 19102 > > Phone: (+) 215-762-7316 > Fax: (+) 215-762-4452 > > Mo Wong wrote: > > I thought I'd post this to the CCP4bb, as > judging by previous posts, it seems I could get > some useful insight into my problem... > > This is question has probably been asked by > people for a long as molecular biology has been > around, but hopefully my question isn't a > complete rehash of other peoples: I am trying to > express a human protein in bacteria where the > only modified amino acids are 3 phosphorylated > serines. I’ve gone through the usual hoopla of > trying to get it expressed in E. coli > (Rosetta/Codon+ cells, varying IPTG, low > temperature, etc). Sequencing confirms my insert > is correct, but from coomassie gel inspection, I > appear to get near zero induction (I need to do > a Western to get a clearer assessment). I’ve > heard about custom gene synthesis, and it > appears Mr. Gene (https://www.mrgene.com/) would > be a good avenue to look into as they optimize > the ORF taking into account codon usage in E. > coli (though I’m not sure they examine > putative mRNA substructure formation like some > companies do). It’s only 49c per base pair, so > doesn’t seem too cost prohibitive. My only > concern is that if this protein is toxic, I > could be wasting money. > > So I was wondering, has anyone seen the > expression for a particular protein change from > zero in Rosetta/Codon+ cells using "native" > sequeneces to being largely overexpressed in > BL21(DE3) cells using codon optimized sequences? > For folks who have had a similar problem to the > one I've described, would you recommend that I > first try using a codon optimized sequence in E. > coli over testing protein expression in > yeast/insect cells, or the other way round? > > Thanks! Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
[ccp4bb] Coot
Hi everybody, I know this is not the coot discussion list but right now I have technical problems with the Internet connection and I could not subscribe it. Here comes my problem. I just installed in a new computer the last distribution package of CCP4 with Coot included. After installation the ccp4 interface runs perfectly but when I tried to run Coot I got this error message: ERROR: In procedure dynamic-link: ERROR: file: "libguile-srfi-srfi-1-v-3", message: "/usr/lib/libguile-srfi-srfi-1-v-3.so.3: wrong ELF class: ELFCLASS64" coot-exe: "/usr/local/bin/xtal/Coot-0.5/bin/coot-real" coot-version: /usr/local/bin/xtal/Coot-0.5/bin/coot-real core: #f No core file found. No debugging I am running Ubuntu 8.10. Thanks a lot in advanced. Ariel
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Like most things (and scientists in general I suppose), SUMO tags have both a good and a naughty side. Like GST, in my hands, they enhanced expression and solubility of a couple of different target proteins. Their primary advantage is that when you clip off SUMO with the SUMO protease (aka Ulp1) they don't leave any unwanted friends behind (i.e. exogenous residues), so you end up with "wild type" protein. The disadvantage is that you need a few barrels of the protease, it is expensive to buy from suppliers (I won't name any names but you know who you are), and the shelf life is about 1-2 months in the -20 freezer . So, we ended up producing it ourselves in E. coli. Good undergrad project :) Also, the enzymatic activity of Ulp1 is very sensitive to [salt] (what's new?) and it is necessary to have NP-40 detergent around. Getting rid of NP-40 is not difficult (ion exchange) but adds an extra step in the purification. Also, I did have issues with the target protein precipitating upon SUMO cleavage. This was solved (normally) by 10% glycerol in the reaction buffer, limiting the time of the reaction, and not letting the protein sit around in a dialysis bag overnight. Once you clip off the SUMO (a His tag is N-terminal to it, so it's clipped, too), just put it back through IMAC and collect the FT. In my experience, it's very clean...no "crud" that I could detect. The proof-in-the-pudding test I guess is crystallizability and I've been able to crystallize two different proteins using this system. Sorry to ramble...hope this helps. Cheers- Brad On Thu, Feb 26, 2009 at 11:30 AM, Phoebe Rice wrote: > We haven't tried SUMO, but had some frustrating results with > GST fusions. They did improve expression and solubility - BUT > in one case the target protein precipitated immediately when > the tag was cleaved off, and resisted all attempts to bring it > back to life. In another case, the fusion protein dragged > chaperones into the prep that were nearly impossible to get > rid of completely, thus ruining our ATPase assays. > > Is SUMO, being smaller, less likely to drag such crud along > with it? > > Phoebe > > > Original message > >Date: Wed, 25 Feb 2009 14:48:57 -0500 > >From: Mo Wong > >Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in > E. coli > >To: CCP4BB@JISCMAIL.AC.UK > > > > Thanks to all who responded. Actually, this bulletin > > board is better for help with molecular biology than > > the molecular biology bulletin board I am subscribed > > to! > > > > On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks > >wrote: > > > > Mo, > > Just to add my 50 cents, I didn't see any > > mention of the use of fusion proteins in your > > original post. GST, MBP or my personal, and > > completely biased, favourite SUMO (plus many more > > proteins) have been shown to enhance expression > > when fused to the amino terminus of a target > > protein. If you fear you have toxicity, simply > > tracking the OD600 pre and post induction normally > > tell you if this is happening. I've worked with > > proteins that basically baselined the cell growth > > upon induction and, as Artem stated, at least I > > knew my protein was being made albeit at very low > > levels. > > > > Stephen > > > > -- > > Stephen Weeks, Ph. D. > > Drexel University College of Medicine > > Department of Biochemistry and Molecular Biology > > Room 10102 New College Building > > 245 N. 15th St. > > Philadelphia, PA 19102 > > > > Phone: (+) 215-762-7316 > > Fax: (+) 215-762-4452 > > > > Mo Wong wrote: > > > > I thought I'd post this to the CCP4bb, as > > judging by previous posts, it seems I could get > > some useful insight into my problem... > > > > This is question has probably been asked by > > people for a long as molecular biology has been > > around, but hopefully my question isn't a > > complete rehash of other peoples: I am trying to > > express a human protein in bacteria where the > > only modified amino acids are 3 phosphorylated > > serines. I’ve gone through the usual hoopla of > > trying to get it expressed in E. coli > > (Rosetta/Codon+ cells, varying IPTG, low > > temperature, etc). Sequencing confirms my insert > > is correct, but from coomassie gel inspection, I > > appear to get near zero induction (I need to do > > a Western to get a clearer assessment). I’ve > > heard about custom gene synthesis, and it > > appears Mr. Gene (https://www.mrgene.com/) would > > be a good avenue to look into as they optimize > > the ORF taking into account codon usage in E. > > coli (though I’m not sure they examine > > putative mRNA substructure formation like some > > companies do). It’s only 49c per base pair, so > > doesn’t seem too cost prohibitive. My only > >
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Some thoughts about SUMO tags and fusion tags in general. Fusion tags also follow the "Garbage In, Garbage Out" philosophy. Yes, if for many of the reasons already hashed out extensively on CCP4BB, one is dealing with lack of expression or miniscule expression, often tagging the protein with a fusion/cleavable tag does indeed bump up the expression and lead to 'improved solubility'. Sometimes, it's very important to ask: improved solubility of what though? Everything that Phoebe describes, namely the chaperone contamination, precipitation after cutting off tag etc., reeks of an intrinsically misfolded/unstable/unhappy protein. My experience-- and those of many others-- is that the fusion tag and fusion tag alone can only fix little in cases: 1) when one observes lots of degradation of the untagged protein, 2) where the untagged protein is made as an intrinsically misfolded/unstable protein. In these cases, the carrier protein then notoriously comes along for the ride in the soluble fraction with the fusion/cleavable tag, initially giving the impression of improved expression and improved solubility. Even then, one might even see multiple degradation products with the tagged expression product. Next, cleave the tag off in such a case and lo and behold! all protein precipitates and you are back to square one. I am not trying to discourage anyone from using fusion tags -- to improve expression, solubility, crystallization etc. We all know of many examples where fusion tags have worked wonders. I only caution that if your favourite protein is intrinsically misfolded in a particular expression system and then you have tried tagging a fusion/ cleavable tag onto the protein in the same expression system and you observe all that Phoebe describes, perhaps it is time to bang your head against a different wall now. In many difficult cases, I am unaware that a fusion tag actually aids in the proper folding of a carrier protein. I will not rule out this possibility but I do not know that this is the general rule. I have worked quite a bit with SUMO tags. As far as GST and SUMO tags are concerned, I banged my head against the GST-tag and SUMO- tag wall for my target protein for a frustrating while. I tried a His tag, then a GST tag, then a SUMO tag. All had exactly the same symptoms. In my case, clearly the problem lay with the carrier problem but I was never allowed to conclude so. Just my two cents, the worth of which will already have diminished by the time you have read this email. Raji On Feb 26, 2009, at 11:30 AM, Phoebe Rice wrote: We haven't tried SUMO, but had some frustrating results with GST fusions. They did improve expression and solubility - BUT in one case the target protein precipitated immediately when the tag was cleaved off, and resisted all attempts to bring it back to life. In another case, the fusion protein dragged chaperones into the prep that were nearly impossible to get rid of completely, thus ruining our ATPase assays. Is SUMO, being smaller, less likely to drag such crud along with it? Phoebe Original message Date: Wed, 25 Feb 2009 14:48:57 -0500 From: Mo Wong Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli To: CCP4BB@JISCMAIL.AC.UK Thanks to all who responded. Actually, this bulletin board is better for help with molecular biology than the molecular biology bulletin board I am subscribed to! On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks wrote: Mo, Just to add my 50 cents, I didn't see any mention of the use of fusion proteins in your original post. GST, MBP or my personal, and completely biased, favourite SUMO (plus many more proteins) have been shown to enhance expression when fused to the amino terminus of a target protein. If you fear you have toxicity, simply tracking the OD600 pre and post induction normally tell you if this is happening. I've worked with proteins that basically baselined the cell growth upon induction and, as Artem stated, at least I knew my protein was being made albeit at very low levels. Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452 Mo Wong wrote: I thought I'd post this to the CCP4bb, as judging by previous posts, it seems I could get some useful insight into my problem... This is question has probably been asked by people for a long as molecular biology has been around, but hopefully my question isn't a complete rehash of other peoples: I am trying to express a human protein in bacteria where the only modified amino acids are 3 phosphorylated serines. I’ve
[ccp4bb] Postdoctoral position ISMB Birkbeck
The School of Crystallography/Institute of Structural and Molecular Biology is seeking a Post-doctoral Research Assistant to carry out structural analysis (x-ray crystallography or cryo-electron microscopy) of complexes formed during pilus biogenesis. The group is led by Professor Gabriel Waksman and has over the years produced numerous high profile publications in the highest impact journals. The research programme is funded by a grant from the MRC to Prof Gabriel Waksman. Further details on the research group can be found at http://people.cryst.bbk.ac.uk/~ubcg54a/ . Applicants should have a PhD in Structural Molecular Biology, Biophysics or a related area, postdoctoral research experience and relevant research publications. Salary range will be from £33,501 to £41,664 per annum inclusive of London Allowance on Grade 7 or 8, initial salary will be dependent on the skills and experience of the successful applicant. Closing date for completed applications: 31 March 2009 To apply for this position please go to www.bbk.ac.uk/jobs and search using reference number 10208.
[ccp4bb] Coot Problem! adding r-nucleotide in DNA
Hi everybody! I am using coot version 0.5. I am trying to build a DNA sequence in my model. At certain positions when i add residues and then used "simple mutate" to make it according to real sequence, it always add ribonucleotide (guanine ribonucleotide, Gr) in place of deoxyribonucleotide (guanine deoxynucleotide Gd). When i say "add residue" it adds adenine deoxynucleotide but on using "simple mutate" function this problem occurs. In rest of the positions in sequence everything works fine except at this particular position. have anybody faced similar problem?? Any suggestion will be appreciated. Thanks in advance. Yusuf
Re: [ccp4bb] another truncate question
Matt Warkentin wrote: > hi folks > > i've almost got my B-factors questions sorted out. one thing i still > don't understand is this: > > in truncate's wilson plot, what is plotted on the vertical axis is not > ln I_mean, but ln (I_mean/ff_mean) > > and ff_mean (or Mn_ff, as truncate calls it) is the "average expected > value of ff". > > my question is how is this value calculated? its resolution > dependence obviously affects the B-factor, so i would like to > understand at least what goes into it. at this point in the analysis, > all truncate knows is the cell/spacegroup, and the approximate number > of AAs right? is this just an expectation of how the amplitudes ought > to fall off vs spacial frequency, without knowing anything about the > detailed structure? The calculation is exactly that. One assumes that each atom scatters X-rays based on its elemental type/charge, that all B factors are equal, and that the atoms are randomly distributed in the unit cell. Since the atoms are not randomly arranged in protein crystals (they having big solvent channels) the curve deviates from linearity at low resolution. It does work pretty good from about 4.5A resolution and higher. If your molecule has some floppy parts and some rigid parts the curve will be a little concave instead of straight. A common mistake is to compare the Wilson B to the average of the B's in your refined PDB file. Because the Wilson B is based on the high resolution end of the plot, atoms with low B factors are given higher weight in the calculation. Whenever your molecule has a spread of B factors, the Wilson B will be lower than the average of the B factors. If you want to make a proper comparison as a check, you should calculate the Wilson B of your Fcalc's and compare that to the Wilson B of your Fobs's. With any properly refined model these two numbers will be nearly identical. In fact, it's a pretty boring check, since I don't know of any program that would fail. > > thanks! Dale
Re: [ccp4bb] how to show double conformation in PyMol
Although there are more elegant ways to do the same, an easy way is to simply write out either just the loop region or the entire molecule containing the second conformation into a second PDB file and to simply read in the two PDB files as two separate molecules in PyMol. Raji On Feb 26, 2009, at 10:10 AM, Yamei Yu wrote: Hi all, There is a 10 residues loop in my structure which has two conformations, how could I show the double conformations at the same time in ribbon or cartoon model in PyMol? Thanks! yamei
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi, I respectfully disagree with the doom&gloom feelings regarding fusion proteins. In my not very limited experience, fusion proteins *can* fix expression issues. Do they always work - of course not :) But there are very few things in this field that work most of the time. Is it better to try a fusion protein or to go into a higher-order expression system? If you can afford it, usually higher order systems tend to work better. But what if you cannot afford it? Regarding precipitation upon cleavage - consider the example of PTPbeta catalytic domain: this protein expresses very poorly on its own, however it expresses extremely well with a His-MBP N-terminal fusion, and the activity of the fusion protein is very high. If you cleave the protein in 'just buffer' then PTPbeta rapidly precipitates. Bad news, right? However if you cleave the fusion in the presence of 0.1% BOG the protein stays perfectly soluble and monomeric, concentrates to 15 mg/ml and produces marvellous crystals (about six structures in the PDB). So - do not be too quick to dismiss fusion proteins as a way to try and salvage your desperate cases, especially if going to a different expression system is hard for some reason. Regarding SUMO - I have personally tested it on about 30-35 proteins. It only worked for *one* - but it made the protein nice and soluble, and it stayed soluble after cleavage (note - we do not use the SUMO-protease, just regular protease sites). Is ratio like that worht the trouble? You decide :) Artem > Some thoughts about SUMO tags and fusion tags in general. > > Fusion tags also follow the "Garbage In, Garbage Out" philosophy. > Yes, if for many of the reasons already hashed out extensively on > CCP4BB, one is dealing with lack of expression or miniscule > expression, often tagging the protein with a fusion/cleavable tag > does indeed bump up the expression and lead to 'improved solubility'. > Sometimes, it's very important to ask: improved solubility of what > though? > > Everything that Phoebe describes, namely the chaperone contamination, > precipitation after cutting off tag etc., reeks of an intrinsically > misfolded/unstable/unhappy protein. My experience-- and those of many > others-- is that the fusion tag and fusion tag alone can only fix > little in cases: 1) when one observes lots of degradation of the > untagged protein, 2) where the untagged protein is made as an > intrinsically misfolded/unstable protein. In these cases, the carrier > protein then notoriously comes along for the ride in the soluble > fraction with the fusion/cleavable tag, initially giving the > impression of improved expression and improved solubility. Even then, > one might even see multiple degradation products with the tagged > expression product. Next, cleave the tag off in such a case and lo > and behold! all protein precipitates and you are back to square one. > > I am not trying to discourage anyone from using fusion tags -- to > improve expression, solubility, crystallization etc. We all know of > many examples where fusion tags have worked wonders. I only caution > that if your favourite protein is intrinsically misfolded in a > particular expression system and then you have tried tagging a fusion/ > cleavable tag onto the protein in the same expression system and you > observe all that Phoebe describes, perhaps it is time to bang your > head against a different wall now. In many difficult cases, I am > unaware that a fusion tag actually aids in the proper folding of a > carrier protein. I will not rule out this possibility but I do not > know that this is the general rule. > > I have worked quite a bit with SUMO tags. As far as GST and SUMO tags > are concerned, I banged my head against the GST-tag and SUMO- tag > wall for my target protein for a frustrating while. I tried a His > tag, then a GST tag, then a SUMO tag. All had exactly the same > symptoms. In my case, clearly the problem lay with the carrier > problem but I was never allowed to conclude so. > > Just my two cents, the worth of which will already have diminished by > the time you have read this email. > > Raji > > > > > > > On Feb 26, 2009, at 11:30 AM, Phoebe Rice wrote: > >> We haven't tried SUMO, but had some frustrating results with >> GST fusions. They did improve expression and solubility - BUT >> in one case the target protein precipitated immediately when >> the tag was cleaved off, and resisted all attempts to bring it >> back to life. In another case, the fusion protein dragged >> chaperones into the prep that were nearly impossible to get >> rid of completely, thus ruining our ATPase assays. >> >> Is SUMO, being smaller, less likely to drag such crud along >> with it? >> >> Phoebe >> >> >> Original message >>> Date: Wed, 25 Feb 2009 14:48:57 -0500 >>> From: Mo Wong >>> Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in >> E. coli >>> To: CCP4BB@JISCMAIL.AC.UK >>> >>> Thanks to all who responded. Actually, this bulletin >>>
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Concerning the contamination of recombinantly expressed proteins with bacterial chaperones, one might wish to try a complete removal using ATP and denatured protein as a "bait" as described here: Protein Expr Purif. 2000 Oct;20(1):45-7. Separation of copurifying GroEL from glutathione-S-transferase fusion proteins. Rohman M, Harrison-Lavoie KJ. Might work or not but could save a lot of time consuming search for the right fusion-tag. Florian Phoebe Rice wrote: We haven't tried SUMO, but had some frustrating results with GST fusions. They did improve expression and solubility - BUT in one case the target protein precipitated immediately when the tag was cleaved off, and resisted all attempts to bring it back to life. In another case, the fusion protein dragged chaperones into the prep that were nearly impossible to get rid of completely, thus ruining our ATPase assays. Is SUMO, being smaller, less likely to drag such crud along with it? Phoebe Original message Date: Wed, 25 Feb 2009 14:48:57 -0500 From: Mo Wong Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli To: CCP4BB@JISCMAIL.AC.UK Thanks to all who responded. Actually, this bulletin board is better for help with molecular biology than the molecular biology bulletin board I am subscribed to! On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks wrote: Mo, Just to add my 50 cents, I didn't see any mention of the use of fusion proteins in your original post. GST, MBP or my personal, and completely biased, favourite SUMO (plus many more proteins) have been shown to enhance expression when fused to the amino terminus of a target protein. If you fear you have toxicity, simply tracking the OD600 pre and post induction normally tell you if this is happening. I've worked with proteins that basically baselined the cell growth upon induction and, as Artem stated, at least I knew my protein was being made albeit at very low levels. Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452 Mo Wong wrote: I thought I'd post this to the CCP4bb, as judging by previous posts, it seems I could get some useful insight into my problem... This is question has probably been asked by people for a long as molecular biology has been around, but hopefully my question isn't a complete rehash of other peoples: I am trying to express a human protein in bacteria where the only modified amino acids are 3 phosphorylated serines. I’ve gone through the usual hoopla of trying to get it expressed in E. coli (Rosetta/Codon+ cells, varying IPTG, low temperature, etc). Sequencing confirms my insert is correct, but from coomassie gel inspection, I appear to get near zero induction (I need to do a Western to get a clearer assessment). I’ve heard about custom gene synthesis, and it appears Mr. Gene (https://www.mrgene.com/) would be a good avenue to look into as they optimize the ORF taking into account codon usage in E. coli (though I’m not sure they examine putative mRNA substructure formation like some companies do). It’s only 49c per base pair, so doesn’t seem too cost prohibitive. My only concern is that if this protein is toxic, I could be wasting money. So I was wondering, has anyone seen the expression for a particular protein change from zero in Rosetta/Codon+ cells using "native" sequeneces to being largely overexpressed in BL21(DE3) cells using codon optimized sequences? For folks who have had a similar problem to the one I've described, would you recommend that I first try using a codon optimized sequence in E. coli over testing protein expression in yeast/insect cells, or the other way round? Thanks! Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp begin:vcard fn:Florian Sauer n:Sauer;Florian org:EMBL-Hamburg;Wilmanns group adr:Notkestr. 85;;C/O DESY bld. 25A;Hamburg;;22603;Germany email;internet:florian.sa...@embl-hamburg.de tel;work:+49 40 89902- 112 version:2.1 end:vcard
Re: [ccp4bb] Coot
Hi, is it possible that you installed for the wrong architecture, i.e., that you have a 32bit machine/OS but picked a 64bit version? It's just a guess (the interface uses tcl/tk so you would not notice from just starting the GUI). Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Thu, 26 Feb 2009, Ariel Talavera wrote: Hi everybody, I know this is not the coot discussion list but right now I have technical problems with the Internet connection and I could not subscribe it. Here comes my problem. I just installed in a new computer the last distribution package of CCP4 with Coot included. After installation the ccp4 interface runs perfectly but when I tried to run Coot I got this error message: ERROR: In procedure dynamic-link: ERROR: file: "libguile-srfi-srfi-1-v-3", message: "/usr/lib/libguile-srfi-srfi-1-v-3.so.3: wrong ELF class: ELFCLASS64" coot-exe: "/usr/local/bin/xtal/Coot-0.5/bin/coot-real" coot-version: /usr/local/bin/xtal/Coot-0.5/bin/coot-real core: #f No core file found. No debugging I am running Ubuntu 8.10. Thanks a lot in advanced. Ariel
[ccp4bb] unit cell flags in mtz files and refmac
Hi all, I am trying to follow good practices and keep my set of free reflections between data sets, eg. in this case between an in-house low resolution and a synchrotron high resolution data set. High resolution data from hkl2000 were imported through the ccp4i task, keeping the low resolution FreeRs. This mtz file contains both unit cells, see below. The refined data set contains only one unit cell description, unfortunately the one from the FreeR (refmac5.5.72 and refmac5.5.70). As the two unit cells are sufficiently different, coot displays the model towards the edge of the density, real space refinement pulls the model back in the middle, refmac then starts with really high Rs and pulls the model back "out". When using rather ancient refmac5.2.0019, the mtz file has the correct unit cell description. Btw. both refinements look about the same. The only difference is a rather annoying shift of the electron density that is displayed in coot based on different unit cell in the mtz file. Is there a way to tell refmac which of the two unit cells to put in the output mtz file? Intuitively, it should be the one from which the amplitudes originate? Cheers Jan *mtz file after import* 1 myprotein high_reso synchrotron 79.0610 79.0610 311.7950 90. 90. 90. 1.0 2 myprotein low_resol rotating_anode 78.5860 78.5860 311.1900 90. 90. 90. 1.54180 *refmac5.5.0072* 2 myprotein low_reso rotating_anode 78.5860 78.5860 311.1900 90. 90. 90. 1.54180 *refmac5.2.0019:* 1 myprotein high_reso synchrotron 79.0610 79.0610 311.7950 90. 90. 90. 1.0 -- -- Jan Abendroth deCODE biostructures Seattle / Bainbridge Island, WA, USA work: JAbendroth_at_decode.com home: Jan.Abendroth_at_gmail.com
Re: [ccp4bb] Coot Problem! adding r-nucleotide in DNA
Hi Yusuf, I had a similar problem once when my pdb file did not have the right format. Check your input coordinate file if your DNA nomenclature is correct for coot. Maybe, the program does not recognize your molecule as DNA. Good luck, christian Yusuf Akhter wrote: > Hi everybody! > I am using coot version 0.5. > I am trying to build a DNA sequence in my model. > > At certain positions when i add residues and then used "simple mutate" > to make it according to real sequence, it always add ribonucleotide > (guanine ribonucleotide, Gr) in place of deoxyribonucleotide (guanine > deoxynucleotide Gd). When i say "add residue" it adds adenine > deoxynucleotide but on using "simple mutate" function this problem occurs. > > In rest of the positions in sequence everything works fine except at > this particular position. > > have anybody faced similar problem?? > > Any suggestion will be appreciated. > > > Thanks in advance. > > > Yusuf > > > > > > > > > -- ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___
Re: [ccp4bb] Coot Problem! adding r-nucleotide in DNA
Thanks to Paul and Christian..for responding very quickly to my query. I tried all versions of coot and it worked in only ver 0.4. Now everything is fine. Cheers! Yusuf On Thu, Feb 26, 2009 at 7:04 PM, Yusuf Akhter wrote: > Hi everybody! > I am using coot version 0.5. > I am trying to build a DNA sequence in my model. > > At certain positions when i add residues and then used "simple mutate" to > make it according to real sequence, it always add ribonucleotide (guanine > ribonucleotide, Gr) in place of deoxyribonucleotide (guanine deoxynucleotide > Gd). When i say "add residue" it adds adenine deoxynucleotide but on using > "simple mutate" function this problem occurs. > > In rest of the positions in sequence everything works fine except at this > particular position. > > have anybody faced similar problem?? > > Any suggestion will be appreciated. > > > Thanks in advance. > > > Yusuf > > > > > > > > > >
[ccp4bb] Questions regarding Peptidoglycan-binding protein
Dear all, Thank you in advance for your expert opinions. I am working on a peptidoglycan (PG)-binding protein, which is composed of two functionally identical domains. Interestingly, when subjected to gel filtration chromatography (Superdex 200), both full-length protein and the single domains migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the expected MW for both samples from the major peak and the shoulder, however, with the presence of smears at high molecular weight. Also, after all fractions from both the major peak and the shoulder were combined and incubated at 4 C over 48 hours, the sample was applied to gel filtration again; this time the shoulder actually disappeared (or shifted), resulting in a nice symmetric peak (with the same elution volume as the major peak). My guess is that the protein expressed in E. coli may already specifically or unspecifically associate with PG fragments from cell wall. My question is: *how to detect PG fragments in my protein sample*? I have tried denaturation-wash-renaturation steps, but I want to know if I have completely got rid of the possible "contaminants". Thank you for suggestions. Joe
[ccp4bb] Purification with ligand
Hello, I wanted to know if there is a standard procedure for purification of protein with ligand. I have never done this before so it will be nice to get some help. Thanks, Mariah -- Mariah Jones Department of Biochemistry University of Florida
Re: [ccp4bb] unit cell flags in mtz files and refmac
Thanks a lot, Garib, as always, there is a new version of refmac just out that fixes all the problems. Cheers Jan 2009/2/26 Garib Murshudov > I think we have fixed this problem just recently. The problem was related > with refmac not being able to pick correct dataset from mtz. IF there was > one dataset in mtz then there was no problem > > Please have a look: > www.ysbl.york.ac.uk/refmac/latest_refmac.html > > Garib > > > On 26 Feb 2009, at 21:52, Jan Abendroth wrote: > > Hi all, > I am trying to follow good practices and keep my set of free reflections > between data sets, eg. in this case between an in-house low resolution and a > synchrotron high resolution data set. High resolution data from hkl2000 were > imported through the ccp4i task, keeping the low resolution FreeRs. This mtz > file contains both unit cells, see below. The refined data set contains only > one unit cell description, unfortunately the one from the FreeR > (refmac5.5.72 and refmac5.5.70). As the two unit cells are sufficiently > different, coot displays the model towards the edge of the density, real > space refinement pulls the model back in the middle, refmac then starts with > really high Rs and pulls the model back "out". > > When using rather ancient refmac5.2.0019, the mtz file has the correct unit > cell description. > > Btw. both refinements look about the same. The only difference is a rather > annoying shift of the electron density that is displayed in coot based on > different unit cell in the mtz file. > > Is there a way to tell refmac which of the two unit cells to put in the > output mtz file? Intuitively, it should be the one from which the amplitudes > originate? > > Cheers > Jan > > > *mtz file after import* > 1 myprotein > high_reso > synchrotron > 79.0610 79.0610 311.7950 90. 90. 90. > 1.0 > 2 myprotein > low_resol > rotating_anode > 78.5860 78.5860 311.1900 90. 90. 90. > 1.54180 > > > *refmac5.5.0072* > 2 myprotein > low_reso > rotating_anode > 78.5860 78.5860 311.1900 90. 90. 90. > 1.54180 > > *refmac5.2.0019:* > 1 myprotein > high_reso > synchrotron > 79.0610 79.0610 311.7950 90. 90. 90. > 1.0 > > > -- > -- > Jan Abendroth > deCODE biostructures > Seattle / Bainbridge Island, WA, USA > work: JAbendroth_at_decode.com > home: Jan.Abendroth_at_gmail.com > > > -- -- Jan Abendroth deCODE biostructures Seattle / Bainbridge Island, WA, USA work: JAbendroth_at_decode.com home: Jan.Abendroth_at_gmail.com
Re: [ccp4bb] unit cell flags in mtz files and refmac
Dear Bart and Howard, let's assume that the paper is novel. Without asking too much, however, I think it would be fair to ask why they used a homology model for molecular replacement, when they claimed successful structure solution two years ago. Also, since they claim successful structure solution, I think it is not too far-fetched to ask them for a preliminary coordinate file for reviewing purposes. Let's give them the freedom of doubt, but let's watch how they respond to the obvious question. I do agree completely with the two of you that in its present form the paper is not acceptable. But if we reject it, they will submit it elsewhere. At least we have the chance of catching something if it is there. Cheers, Manfred. On Thu, 26 Feb 2009, Jan Abendroth wrote: Thanks a lot, Garib, as always, there is a new version of refmac just out that fixes all the problems. Cheers Jan 2009/2/26 Garib Murshudov I think we have fixed this problem just recently. The problem was related with refmac not being able to pick correct dataset from mtz. IF there was one dataset in mtz then there was no problem Please have a look: www.ysbl.york.ac.uk/refmac/latest_refmac.html Garib On 26 Feb 2009, at 21:52, Jan Abendroth wrote: Hi all, I am trying to follow good practices and keep my set of free reflections between data sets, eg. in this case between an in-house low resolution and a synchrotron high resolution data set. High resolution data from hkl2000 were imported through the ccp4i task, keeping the low resolution FreeRs. This mtz file contains both unit cells, see below. The refined data set contains only one unit cell description, unfortunately the one from the FreeR (refmac5.5.72 and refmac5.5.70). As the two unit cells are sufficiently different, coot displays the model towards the edge of the density, real space refinement pulls the model back in the middle, refmac then starts with really high Rs and pulls the model back "out". When using rather ancient refmac5.2.0019, the mtz file has the correct unit cell description. Btw. both refinements look about the same. The only difference is a rather annoying shift of the electron density that is displayed in coot based on different unit cell in the mtz file. Is there a way to tell refmac which of the two unit cells to put in the output mtz file? Intuitively, it should be the one from which the amplitudes originate? Cheers Jan *mtz file after import* 1 myprotein high_reso synchrotron 79.0610 79.0610 311.7950 90. 90. 90. 1.0 2 myprotein low_resol rotating_anode 78.5860 78.5860 311.1900 90. 90. 90. 1.54180 *refmac5.5.0072* 2 myprotein low_reso rotating_anode 78.5860 78.5860 311.1900 90. 90. 90. 1.54180 *refmac5.2.0019:* 1 myprotein high_reso synchrotron 79.0610 79.0610 311.7950 90. 90. 90. 1.0 -- -- Jan Abendroth deCODE biostructures Seattle / Bainbridge Island, WA, USA work: JAbendroth_at_decode.com home: Jan.Abendroth_at_gmail.com -- * * *Dr. Manfred S. Weiss * * * * Team Leader * * * * EMBL Hamburg OutstationFon: +49-40-89902-170 * * c/o DESY, Notkestr. 85 Fax: +49-40-89902-149 * * D-22603 Hamburg Email: mswe...@embl-hamburg.de * * GERMANY Web: www.embl-hamburg.de/~msweiss/ * * *
Re: [ccp4bb] Purification with ligand
Hi Mariah, You may need to specify what type of ligand (is it a nucleotide, a small synthetic molecule, a peptide etc ?) and also what is the affinity between your ligand and your protein. I have purified several protein-ligand complexes, you can go several routes. If you have a high affinity binder and fairly 'cheap' or abundant ligand it is possible for example to add it in your crude extract (or even in your bacterial culture in some extreme cases). It may end-up improving your 'expression yield' in terms of soluble protein readily available in your initial extract. I have used this approach successfully when purifying the ligand-binding domain of a nuclear receptor with its very hydrophobic natural ligand (retinoic acid) or a synthetic drug. In this case it was giving a more homogenous population of protein at the start of the purification. I included the hormone in the crude extract after sonication and centrifugation (so at an early stage of the purification). After that I kept ligand around in the buffer (Cobalt affinity chromatography and gel filtration (at low concentration) and kept adding ligand to the concentrated protein. If your ligand has some kind of specific UV absorption, it can be very easy to monitor its presence and the 'saturation level' of your protein. If you have a high-affinity binder, it can be a very efficient way to start with homogenous population of protein-ligand complex. This approach is really useful when your ligand happens to be only soluble in protein-unfriendly solvents like ethanol or acetone (this was the case for retinoic acid); in the crude extract despite the addition of alcohol, your protein won't suffer too much from the presence of added alcohol and if affinity is high and you add enough of it, you will efficiently saturate it. In another case, a complex between two GTPases, I had to use a non-hydrolyzable GTP analog. The compound was far too expensive to be used in the crude extract. In this case , we purified the two apo-proteins separately, formed the complex in presence of ligand and included ligand in the ion-exchange chromatography buffers and in the gel filtration buffer (at a low concentration though but it helped us to stabilize the complex). Again it all depends on the affinity.If you decide to include ligand in your gel-filtration buffer, keep also in mind that you will contaminate your columns and it can be hard to get rid of some ligands sometimes. Sorry, if all this was a little bit too long. Hope this helps, Pascal Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics Laboratory of Robert Stroud On Thu, Feb 26, 2009 at 2:58 PM, protein.chemist protein.chemist < pp73...@gmail.com> wrote: > Hello, > I wanted to know if there is a standard procedure for purification of > protein with ligand. I have never done this before so it will be nice to > get some help. > > Thanks, > Mariah > > -- > Mariah Jones > Department of Biochemistry > University of Florida >
Re: [ccp4bb] Purification with ligand
Hi, I wouldn't call this a standard procedure - generally speaking the most important two parameters you have to consider are: the protein/ligand affinity and the supply of the ligand. For tightly bound ligands, you may be able to just add the ligand once (i.e. during cell growth and expression, or upon lysis) thus avoiding the need to add the ligand to the chromatographic buffers throughout the process. This is perfect for ligands that aren't available in large quantities. The flip side of this is that when you're done purifying you're kind of stuck with the ligand you have - unless you successfully exchange it for something else (which is often hard to do completely if ligand has single digint nanomolar Ki or lower). On the other end of the spectrum are ligands that bind not as tightly and can be readily exchanged - these require you to have the ligand everywhere in the chromatography in the amount sufficient to elicit nearly full occupancy of the active site. Obviously it's better if you have such ligands in good supply. Examples of the tight-ligand co-purification are: GPCRs, nuclear hormone receptors (PPAR/RXR, RAR/RXR, etc.) - these proteins often require ligands for stability; and certain kinases that are otherwise toxic to the cells (PKC family) and have to be expressed/purified with a ligand because otherwise they kill the expression host. Examples of the medium- or weakly- bound ligand co-purifications include many nucleotide/side acting enzymes, aminotransferases (often need PLP to remain active and stable), proteins that require specific metal ions to remain in good shape, and a number of proteases that tend to chew themselves up unless they have an inhibitor to gnaw on. Sometimes ligands are co-purified unintentionally (famous example - quorum sensing factor with the crazy boron chemical) and pop up in the electron density as a surprise to the crystallographer. Please don't hesitate to ask specific questions :-) Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of protein.chemist protein.chemist Sent: Thursday, February 26, 2009 5:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Purification with ligand Hello, I wanted to know if there is a standard procedure for purification of protein with ligand. I have never done this before so it will be nice to get some help. Thanks, Mariah -- Mariah Jones Department of Biochemistry University of Florida
Re: [ccp4bb] Questions regarding Peptidoglycan-binding protein
Look up a standard endotoxin assay (usually an immunoassay) because that's exactly what endotoxin is :-) Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of JOE CRYSTAL Sent: Thursday, February 26, 2009 5:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Questions regarding Peptidoglycan-binding protein Dear all, Thank you in advance for your expert opinions. I am working on a peptidoglycan (PG)-binding protein, which is composed of two functionally identical domains. Interestingly, when subjected to gel filtration chromatography (Superdex 200), both full-length protein and the single domains migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the expected MW for both samples from the major peak and the shoulder, however, with the presence of smears at high molecular weight. Also, after all fractions from both the major peak and the shoulder were combined and incubated at 4 C over 48 hours, the sample was applied to gel filtration again; this time the shoulder actually disappeared (or shifted), resulting in a nice symmetric peak (with the same elution volume as the major peak). My guess is that the protein expressed in E. coli may already specifically or unspecifically associate with PG fragments from cell wall. My question is: how to detect PG fragments in my protein sample? I have tried denaturation-wash-renaturation steps, but I want to know if I have completely got rid of the possible "contaminants". Thank you for suggestions. Joe
[ccp4bb] What can a learning student should post in our forum- are any restrictions for discussing science[what is science?] with intellectuals :)
Dear scientists I have asked a question in previous discussions regarding cryo conditions for collecting data of a crystal. What is the cryo condition followed in a small molecule data collection in olden days. I got wonderfull answers.i am very happy for that. Is any one confused in this issue like me. Let me know more questions regarding this issue. And more over i am criticised that it is not a good thing that i said i am confused in this issue by some of the researchers. And i have been mentioned that i should not point the concept of confusion in our forum. Well what is confusion means in this world of research ? Any thing which is not understood is confusion? or Any thing that is not known is confusion? If we dont know the answer wether it is yes or no then it is confusion? Can we use the word confusion in the field of research ? I am still confused with what is right and wrong in this world of research where the point of reference changes with time and condition. Some body made me to accept that science and philosophy are verymuch different?Are they ? S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany.
Re: [ccp4bb] protein folds
On Feb 25, 2009, at 11:45 AM, Jayashankar wrote: But provided if i colud follow a pattern even blindfolded could come up with some probable things. Is it not ok to have principle of mathematical induction. Or extrapolation. I would make a plot of new folds discovered by year and extrapolate into the future. I might use the variance in the fit as some indication of uncertainty.
[ccp4bb] Centramate
Hi, Sorry for the off topic question. I am looking for helps from someone who is familiar with Centramate from Pall. We recently bought one and try to use it for concentrating insect media with secreted proteins. Here is some more information: Pump: MasterFlex Console drive Pump head: quick load, model 7021-26 Tubing: We originally use PharMed L/S24. However, we quickly realized that the pump head will cut the tube. We then switched to PharMed L/S15. Membrane: We use an Omega cassette with 10k cutoff. Our protein is about 25 kDa. We use Hyclone SFX-insect media. The supernatant is pre-filtered using a 0.45 um membrane before being loaded to Centramate. We add a pressure at about 20 psi with about 8 psi backpressure. It is quite fast at the beginning and then turns slower and slower. For the experiment I just did today, it took about 5 hours to go from 2L down to about 300 ml. This is ridiculous! Did I do something wrong? The tech at Pall insisted that it must be due to some strange ingredient in the Hyclone media. Anyone agree? Any common will be highly appreciated. Best, Chen
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi all, Once again I seem to have managed to kick up a minor debate on the bulletin board (Note to self no more posts on SUMO or Apple :-[ ). With quite a few years of experience working with SUMO I feel I can safely state that it is a good enhancer of fusion protein production in E. coli. I am personally NOT convinced that it is a "solubility" enhancer like MBP or NusA but the fusions main benefit is it's easy and specific removal. By default I do 24oC inductions in Bill Studiers fantastic auto-inducing media so I haven't really fought with solubility issues for a while. We make and use our own hydrolase in the absence of any detergent (unless of course it the target protein requires it) , and I find it better than TEV and PreScission (admittedly I have not tried the new more soluble clones of the former). Typically we get 100 mg/L using autoinduction media which we dilute to 0.5 mg/ml in 50% glycerol buffer, salt and DTT, of which I'll use 100-200 ul for a fusion protein prep of 100 mg plus. Addressing Mo's original question I shall restate my answer as: that it would be cheaper to stick his construct into any fusion vector he can lay his hands on before handing money over to the gene synthesizers to see if he can get detectable _expression_. Thinking downstream, if it works, you need to consider the expense of the removal of the fusion partner. Clones are available for TEV, PreScission (Rhinovirus 3c protease) and of course SUMO hydrolase ;-) (plus there are few systems out there for removal of tags without a protease). No single fusion system is a panacea for all our protein _expression_ woes and stating a position on one is equivalent to choosing sides in the Mac vs. PC debate. (Actually I have an idea for an advert featuring SUMO, the small and hip fusion partner and MBP, the dull old and overweight workhorse). Stephen ar...@xtals.org wrote: Hi, I respectfully disagree with the doom&gloom feelings regarding fusion proteins. In my not very limited experience, fusion proteins *can* fix _expression_ issues. Do they always work - of course not :) But there are very few things in this field that work most of the time. Is it better to try a fusion protein or to go into a higher-order _expression_ system? If you can afford it, usually higher order systems tend to work better. But what if you cannot afford it? Regarding precipitation upon cleavage - consider the example of PTPbeta catalytic domain: this protein expresses very poorly on its own, however it expresses extremely well with a His-MBP N-terminal fusion, and the activity of the fusion protein is very high. If you cleave the protein in 'just buffer' then PTPbeta rapidly precipitates. Bad news, right? However if you cleave the fusion in the presence of 0.1% BOG the protein stays perfectly soluble and monomeric, concentrates to 15 mg/ml and produces marvellous crystals (about six structures in the PDB). So - do not be too quick to dismiss fusion proteins as a way to try and salvage your desperate cases, especially if going to a different _expression_ system is hard for some reason. Regarding SUMO - I have personally tested it on about 30-35 proteins. It only worked for *one* - but it made the protein nice and soluble, and it stayed soluble after cleavage (note - we do not use the SUMO-protease, just regular protease sites). Is ratio like that worht the trouble? You decide :) Artem Some thoughts about SUMO tags and fusion tags in general. Fusion tags also follow the "Garbage In, Garbage Out" philosophy. Yes, if for many of the reasons already hashed out extensively on CCP4BB, one is dealing with lack of _expression_ or miniscule _expression_, often tagging the protein with a fusion/cleavable tag does indeed bump up the _expression_ and lead to 'improved solubility'. Sometimes, it's very important to ask: improved solubility of what though? Everything that Phoebe describes, namely the chaperone contamination, precipitation after cutting off tag etc., reeks of an intrinsically misfolded/unstable/unhappy protein. My experience-- and those of many others-- is that the fusion tag and fusion tag alone can only fix little in cases: 1) when one observes lots of degradation of the untagged protein, 2) where the untagged protein is made as an intrinsically misfolded/unstable protein. In these cases, the carrier protein then notoriously comes along for the ride in the soluble fraction with the fusion/cleavable tag, initially giving the impression of improved _expression_ and improved solubility. Even then, one might even see multiple degradation products with the tagged _expression_ product. Next, cleave the tag off in such a case and lo and behold! all protein precipitates and you are back to square one. I am not trying to discourage anyone from using fusion tags -- to improve _expression_, solubility, crystallization etc. We all know of many examples where fusion tags have worked wonders. I only caution that if your
Re: [ccp4bb] Questions regarding Peptidoglycan-binding protein
Apologies for the slightly frivolous language - technically peptidoglycan is *an* endotoxin, as there's a separate entity also commonly referred to as endotoxin which is the LPS/LOS component of the cell wall. Peptidoglycan is also an endotoxin (or at least quite a few researchers consider it to be one) which is why I would like to correct my previous answer. You could try BacTx from Immunetics, for pure peptidoglycan assay. Not sure if it's fully commercial or not but they might send you a sample. Since endotoxin contamination and peptidoglycan contamination usually go hand in hand, the endotoxin assay should be indicative of PG presence as well. The crab blood assay (I am not kidding!) is probably too sensitive as it is designed to detect minuscule amounts of contamination. Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: Artem Evdokimov [mailto:ar...@xtals.org] Sent: Thursday, February 26, 2009 6:57 PM To: 'JOE CRYSTAL' Cc: 'CCP4BB@JISCMAIL.AC.UK' Subject: RE: [ccp4bb] Questions regarding Peptidoglycan-binding protein Look up a standard endotoxin assay (usually an immunoassay) because that's exactly what endotoxin is :-) Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of JOE CRYSTAL Sent: Thursday, February 26, 2009 5:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Questions regarding Peptidoglycan-binding protein Dear all, Thank you in advance for your expert opinions. I am working on a peptidoglycan (PG)-binding protein, which is composed of two functionally identical domains. Interestingly, when subjected to gel filtration chromatography (Superdex 200), both full-length protein and the single domains migrate as a major peak tailed with a broad shoulder. SDS-PAGE indicates the expected MW for both samples from the major peak and the shoulder, however, with the presence of smears at high molecular weight. Also, after all fractions from both the major peak and the shoulder were combined and incubated at 4 C over 48 hours, the sample was applied to gel filtration again; this time the shoulder actually disappeared (or shifted), resulting in a nice symmetric peak (with the same elution volume as the major peak). My guess is that the protein expressed in E. coli may already specifically or unspecifically associate with PG fragments from cell wall. My question is: how to detect PG fragments in my protein sample? I have tried denaturation-wash-renaturation steps, but I want to know if I have completely got rid of the possible "contaminants". Thank you for suggestions. Joe
Re: [ccp4bb] screensaver
Just a quick reminder, those of you who forgot about the "deadline" for submitting some nice crystallography related pictures it will be extended until March 9th. I've received some really nice pictures and rendered images so far, keep sending more. 2 per person, just as a reminder. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Biochemistry and Molecular Biology, W8708 615 North Wolfe Street Baltimore, MD 21205 Phone: +1-410-614-4742 Fax: +1-410-955-3655
