[ccp4bb] Difficult purification with imac columns
Hi. We are working on a periplasmic protein that breaks naked glycans in peptidoglycans. There is truncated structure available but our target is the full length protein. The difficulty us that it strongly binds to the resin with or without his.tag. Changing the resin to acrylamide did not help. Has anyone come across similar problem and how was it resolved. The pdb structure is the catalytic domain and mussing a region that, in my opinion, binds to the resin. Thank you in advance Sent from my iPhone
Re: [ccp4bb] conversion of IU/ml to mcg
On 1/19/12 5:32 AM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Megha, your email could hardly be more cryptic to me, and maybe you increase the chance of getting help by explaining - - what is R D - - what is MiOU? - - what is mcg? (milli-centi-gram?) (I understand ml, but I do not remember having met any of those other units). Cheers, Tim On 01/19/2012 03:58 AM, megha goyal wrote: We are involved in R D of recombinant filgrastim and the standard sample label mentions it as 30MiOU/ml i.e 300 mcg/ml. How can we determine the IU/ml as we know our protein is 300 mcg/ml. can anyone please guide me on the corelation. regards, megha - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPF/EtUxlJ7aRr7hoRAk6VAKDL7l0sN0R5PigjSd8cfA3bKB94lwCfSM8i zfVXB/IzdZnHJrI/0xj4aUY= =X/M9 -END PGP SIGNATURE- Would these be RD - Research and Development? mcg = microgram IU/ml = International Units?/ml I have no idea about MiOU? Probably some optical unit? Subbu
[ccp4bb] Paper describing the structure of LFA-1
Dear All: I would like to know the literature on the crystal structure of Leukocyte Function-Associated Antigen One (LFA-1, CD11a/CD18). I have the structure of I domain but not for the entire molecule. I would greatly appreciate if people can point me to the right article/reference. Thanks Subbu
[ccp4bb] Concentrating a protein solution - subbu
Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that others use to increase the concentration other that using amicon columns. Any help will be appreciated. Thanks Subbu
[ccp4bb] In XDS, anomalous correction column has negative values
Dear All: What does it mean when I get negative values under Anomalous Corr column after running XDS? I set the Friedel Law=False even though I suspect that my signal is very very weak. Thanks Subbu
[ccp4bb] Non-CCP4 protein purification problem - help needed
Hi all: I am trying to purify a protein that was expressed in a baculovirus system. The protein elutes in the flow through in a DE52 column. Further gel filtration gives an enriched protein but we see that some medium components tag along (weight and activity data do not match). Another mutant in the series also gives 30 mg of colorless fluffy material but again we never got this much protein for other mutants in the same series. We suspect that some medium is tagging. We tried other chromatography such as hydroxyapatite, activated charcoal, anion exchange etc but not successful in getting rid of the impurity. Has anyone come across a protein that eluted in the flow through and if so, what is their experience? Did they notice such behavior? Your advice will be greatly appreciated. Thanks Subbu
Re: [ccp4bb] Per-residue RMSD for multiple structures?
Gerard DVD Kleywegt wrote: Thanks Stephen! I was going to suggest that, but I was afraid of the self-appointed CCP4BB Gestapo that has been seen goose-stepping in this neighbourhood recently (Tassos recently accused me of becoming mellow and diplomatic in my dotage, so I hope I've set the record straight now). However, since this solution is neither CCP4 nor Phenix, we may get away with this heinous act of bulletin-board heresy... On the other hand, I've learned that it is often more expedient to beg for forgiveness than to ask for permission. I would add that: - I assume that the sequences and numbering are identical - you should put the structures in one big PDB file and read it into LSQMAN - since LSQMAN doesn't do true multiple-structure alignment, you could pre-align them, e.g. with SSM/PDBeFold - if you didn't, you could indeed use the MCentral and MAlign commands to align them - my favourite plot would be the CD plot (but then again, it would, wouldn't it?) - see for instance: http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/cdplot_1ldn.gif - which is also produced with the MPlot command - http://xray.bmc.uu.se/usf/lsqman_man.html#S82 - a normal MPlot would look like this: http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/mplot_1ldn.gif - the output file of the normal MPlot command is in a form that can be quickly converted into an O datablock for those handy with an editor and familiar with O datablocks, and could then be used to ramp a model inside O - you may also want to consider showing how the (main-chain or side-chain) torsion angles differ between the structures, e.g. by plotting the circular variance of phi and psi - see for instance http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/vmain_1ldn.gif - as described here: http://xray.bmc.uu.se/usf/lsqman_man.html#S83 - or a multiple-model Ramachandran plot like this http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/mrama_1ldn.gif (with the MRama command). The advantage is that no superposition is required at all and that any domain movements won't debeautify your results --dvd On Tue, 23 Feb 2010, Stephen Graham wrote: I am pretty sure you can do this using LSQMAN from Gerard (BluRay?) Kleywegt. The pertinent commands are MCENTRAL to determine the 'most representative structure' (i.e. the one to align upon and show in the figure), MALIGN to do the alignment and then MPLOT to calculate a 'multi-RMSD' for each residue (see manual for details - set the 'cut-off for printing' to 0 to get all values). Regards depiction, I think pymol can also represent structures as sausages based on their B values: cartoon putty show cartoon HTH, Stephen On 23 February 2010 01:31, Ethan Merritt merr...@u.washington.edu wrote: Hi all, I am comparing 4 very similar (1.5A rmsd) large (750 residues) structures, but struggling to find a way to generate a figure that conveys where they are most alike and where they diverge. Simply drawing a superimposed set of backbone traces results in what looks like colored spaghetti. I don't think that's going to work. So I had the idea of drawing a single backbone trace, or ribbon diagram, and coloring by the RMSD of the four C-alphas at each residue position. But I can't find a program that will output this as a table of numbers I can use. All of the multiple structure superposition programs must have this information internally. After all, that's what they are minimizing. But do any of the programs provide an option to write it out? I can get pairwise per-residue deviations by doing SSM superposition in Coot, but that doesn't get me to an RMSD for all four structures jointly. Ethan -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742 -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638 Best wishes, --Gerard ** Gerard J. Kleywegt Dept. of Cell Molecular Biology University of Uppsala Biomedical Centre Box 596 SE-751 24 Uppsala SWEDEN http://xray.bmc.uu.se/gerard/ mailto:ger...@xray.bmc.uu.se ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. ** May be if you changed your name to BlueRay, you can get away with this heresy. Subbu
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
Clemens Vonrhein wrote: Hi Jim, On Fri, Jan 22, 2010 at 09:11:00AM -0600, Jim Pflugrath wrote: I do not know the effect myself, but the idea of vibration fee has been tossed around a while. What? I'll have to pay for vibration now as well? Are you collecting that personally? Or is it just another scheme to save some failing banks? Maybe I'm lucky and that only applies to US citizens ... I'll ask my tax adviser ... Clemens Only if you exercise! Subbu
[ccp4bb] Error in CAD during ARP/wWARP run in sharp/autoSHARP - also posted in sharp discuss
Hi: Sorry for the same posting in here as well but I thought may be some of you might have encountered the same problem but may not be subscribing to sharp list. I am trying run sharp/autoSHARP on a mac (os x 10.4) using the guven example file: krel1-SAD.0 I am getting an error at the CAD as provided below ++ The parameter file is: /Users/subbu/sharp/sharpfiles/logfiles_local/krel-SAD.1/wARP_49.4pc/20091213_080749/arp_warp_tracing.par Entering warp_tracing.sh from the command line The working directory is: /Users/subbu/sharp/sharpfiles/logfiles_local/krel-SAD.1/wARP_49.4pc/20091213_080749 ARP/wARP will run in the subdirectory: temp_tracing Building free atoms model Initial map will be calculated with pre-weighted amplitudes ... CAD: Error in label assignments in LKYSET QUITTING ... ARP/wARP module stopped with an error message: CAD ++ The relevant CAD log file is also attached below Chosen Asymmetric unit of reciprocal space: [mmm] hkl:h=0, k=0, l=0 ** Missing flag set in HKLIN1 to Nan: ** Missing entries LISTED as -999.000 Data line--- LABIN E1=FBshasol E2 MtzParseLabin: run out of labels trying to match E2 CAD: Error in label assignments in LKYSET ++ This happened on another dataset so I wanted to see whether the example file runs ok. Please help Subbu
[ccp4bb] Difficult MR structures
Deal All: I have a 2.0 A data for a SeMet protein (native crystal not available yet!) that has 6 Se sites. The cell comes out to be 65 67 101 and the angles are all very close to 90. The data set was collected in house with Cu 1.5418 A We integrated and scale in orthorhombic and the statistics are reasonable. There are 4 homologous structures and all of them have a sequence identity of 15-16 %. I have tried Phaser, AMoRe, EPMR with varying templates with polyala, polyser and varying the resolutions. I have also done a modeller and found best match with one of the four structures. I have used this derived model as well for input. I would like to know whether there other options (I am working on getting a good synchrotron data set at the peak wavelength for Se). Thanks Subbu PS: I am also trying P1 just in case.
[ccp4bb] AU limts not read - arp/warp ccp4i interface
Hi: I tried to run arp/warp using the gui. My original mtz file (space group p222) is read correctly and I see the AU limits in the Crystal parameters. I then tried to change the space group using CAD/SORTMTZ export to .sca and manually change the space group to P2212 and then used scalepack2mtz to generate an mtz file. The space group is correctly converetd and the SYMM is fine. But arp/warp comes up with AU limits error. Please help. Subbu
[ccp4bb] Memory allocation error in phaser - Mac os
Dear All: How to increase the memory size (?) to avoid the following error? *** * Information from CCP4Interface script *** The program run with command: /sw/share/xtal/ccp4-6.1.1/bin/phaser has failed with error message phaser(1393) malloc: *** vm_allocate(size=402313216) failed (error code=3) phaser(1393) malloc: *** error: can't allocate region phaser(1393) malloc: *** set a breakpoint in szone_error to debug phaser(1393) malloc: *** vm_allocate(size=402313216) failed (error code=3) phaser(1393) malloc: *** error: can't allocate region phaser(1393) malloc: *** set a breakpoint in szone_error to debug *** Thanks Subbu
[ccp4bb] How to compile a small fortran program which uses some CCP$ modules in fortran
Hi: I have an old fortran code that I used on an SGI Irix system. i would like to use it on a linux (Ubuntu). How to compile this code which uses some ccp4 libraries? What is the command for this? Thanks Subbu
Re: [ccp4bb] video that explains, very simply, what Structural Molecular Biology is about
mb1pja wrote: Dear Fred A really nice video that would be great for giving non-crystallographers (including colleagues and 1st year students, and perhaps also friends and family) an overview of what we do. Thank you for pointing it out - and of course very many thanks to Dominique Sauter for making it. I am sure it will prove very popular. bet wishes Pete (Pete Artymiuk) On 11 Nov 2009, at 09:44, Vellieux Frederic wrote: Dear all, Thought I'd share this with you: I located this through Ms Ines Kahlaoui, from the Beja Higher Institute of Biotechnology in Tunisia (Ines has to teach and locates videos on the internet, which she then downloads and uses for teaching). Ines located this jewel: http://video.google.com/videoplay?docid=7084929825683486794ei=M3b5SvXqD6em2AK3jY33CQq=Plongee+coeur+vivant# This is the French version (explains everything about Structural Molecular Biology, but for the maths :-( , but also shows what we crystallographers have known for a long time, since the first colour ES graphics workstations in fact, that the electron are blue :-) ). Both French and English versions can be downloaded from http://cj.sauter.free.fr/xtal/Film/ No rights associated with the movie, and the Strasbourg group intends to release a higher quality version on DVD soon. Please contact them about that... I am only sharing what I thought was good for educational purposes. 18 minutes of your life, but worth it I think. So feel free to share this. Wish you all a nice day, Fred. Hi: Could someone point out the name and where to get these crystallization plates used in the video? By the way, this is a wonderful video. Subbu
[ccp4bb] [Fwd: Re: [ccp4bb] video that explains, very simply, what Structural Molecular Biology is about]
Just thought this will be of interest to all. Subbu ---BeginMessage--- Narayanan Ramasubbu wrote: Vellieux Frederic wrote: Narayanan Ramasubbu wrote: Hi: Could someone point out the name and where to get these crystallization plates used in the video? By the way, this is a wonderful video. Subbu Hi Subbu, If you order these plates (EasyXtal Tool X-Seal from QIAGEN), there are 2 types: white O rings, like those shown in the video (evaporation through the O ring) and black O rings (less evaporation though the ring so that the crystallisation droplets last longer). HTH, Fred. Thank you. Subbu Don't mention it. If you think this is useful information, feel free to forward the part of the mail that is concerned with the O rings to ccp4bb. Have a nice weekend, Fred. ---End Message---
[ccp4bb] Post-doctoral position available June 1st at UMDNJ
A postdoc position is immediately available in my laboratory at the Oral Biology Department, UMDNJ. The post-doc will work on the elucidation of structure of enzymes involved in modifying A. actinomycetemconitans biofilms. Crystals of two such enzymes are available. Experience in expression and purification using his tag is essential. Additional experience in yeast expression is desirable. A highly motivated individual with at least one year experience in structural biology and protein biochemistry who is a team player is sought. This position is for one year with the potential for additional years depending upon funding. Please send your CV and names of three references to Dr. N. Ramasubbu (ramas...@umdnj.edu). Subbu
Re: [ccp4bb] Structural biology inside the cell
ar...@xtals.org wrote: Hello, 1. As long as all proteins have seventy amino-acids or less and express in E. coli in mM concentrations - we're in business. 2. As for the question below - my favorite answer is 'It will take a week and ten million dollars in unmarked bills. We begin as soon as the money arrives.'. Artem As another example, more than once I have been asked by someone if they give me the sequence or name of a protein (even membrane proteins), how many days would it take us to provide them the crystal structure. Mark Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ On 12 Mar 2009, at 21:57, Felix Frolow wrote: Dear Mark Stay calm Buzz-words come and very frequently do not stay, they go away with the artifacts they advocate... Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Mar 12, 2009, at 6:14 PM, Mark J. van Raaij wrote: Dear All, a News Views article in Nature 458, pages 37-38 of 5 March 2009 (link below) states: The development of structural biology WAS historically based on the principle of divide and conquer — individual proteins were purified to homogeneity and their atomic structures were solved in vitro by using either X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. This approach WAS tremendously successful, and led to the creation of a protein-structure databank that currently contains more than 50,000 structures. I find the past tense here too much... Greetings, Mark Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ http://www.nature.com/nature/journal/v458/n723 4/full/458037a.html Structural biology: Inside the living cell And the work will be carried out in the Caribbean... Subbu
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
ar...@xtals.org wrote: Hi, In a case like the one Raji outlined below - after all the attempts - I would have most cerainly switched to insect cells as the next step :) If you suspect that protein of interest has large disordered regions, expression in a higher order system by itself may not be enough (still good chances of proteolysis). I am guessing here, but co-expression with a partner is probably necessary (as per your point #4). All of this assumes that you need the entire protein, of course (as Raji said) - because otherwise the fairly obvious next step could be to chop that beast into manageable chunks :) Is this 'normal' and 'expected'? I would say that yes, if you are working with a large human protein that has potentially disordered regions and is normally a part of a stable complex with other stuff - yes you can expect problems exactly like these (or worse). Artem BUT...I recently had a similar problem to what Mo outlines -- human protein in E. coli, 'zero induction', wondering if the protein is toxic based on colony morphology and effect on cell growth, and all the good stuff that Mo describes. In my case, for my human protein: 1. Disorder-prediction and secondary-structure-prediction programs indicate stretches of disordered protein 2. After the usual tricks of bacterial expression with a His-tagged version (temp, IPTG variation, autoinduction, cells), I got 'zero induction'. Then I switched to larger fusion tags like those both of you mention and then I could see expressed protein, though there was only a small fraction of full-length protein and copious amounts of proteolysis and/or truncated products (mass spec, Western blot etc. etc.). 3. When one tag didn't work, I switched to another. After switching a bunch of tags, I consistently faced the same kinds of problematic issues like persistent proteolytic degradation, persistent aggregation even when fusion tag was left intact, and hard-to-remove chaperone contamination during purification. I was unable to recover purified full-length protein and these issues could not be mitigated by buffer, pH optimization etc. during purification etc. 4. Literature indicates that the protein might function in a complex In my case, I used all of the above information collectively to hypothesize that my target protein, under the given conditions, was likely poorly folded on its own. In this specific case, even before I determined whether to move to a different expression system or not, I did still have at least a few choices that I can think of: a. To attempt co-expression with a protein binding partner b. To attempt co-expression in a chaperone-aided expression system c. To resort to a synthetic gene (Chopping up the protein and going after individual domains was not a real option in my case.) Is my example the norm or the exception? I leave that open. Even if my case is extremely rare, my original intention was to caution that there may come a time with a beast of a protein-- after a LOT has been attempted and tried-- when one has to start to quickly distinguish between a promising approach and a futile time-sink. To me, it seems we concur and there is not much scope for a healthy debate :)! Raji On Feb 26, 2009, at 10:07 PM, Artem Evdokimov wrote: I personally think that debate is healthy. Having said this – I do not advertise one fusion partner over another – and I did not intend to overweight the MBP success in my previous message, it was just a familiar example. As I mentioned, SUMO also worked for us – perhaps not as frequently as MBP but there is no statistical significance in this because the number of studied cases was way under a thousand (which intuitively sounds like a useful number, but in fact even a thousand is probably too small to be meaningful). Unlike choosing one brand of OS over another, the choice of fusion partner (or indeed whether to use or not to use one) is not an expensive one since cloning is generally very cheap and expression in E. coli is also extremely affordable. Therefore it is perfectly easy to try as many expression tricks as one desires. Having said this I also should mention that enough trials in E. coli eventually out-price a few trials in insect cells or other ‘higher order’ system so everyone should apply their own judgement as to when the aggregate cost advantage disappears. I don’t think that anyone would disagree that chances of successful expression of a eukaryotic protein in e.g. insect cells are considerably higher compared to the same odds in bacteria, but unfortunately so is the price both in terms of money and of labor. Removal of fusion tags is a separate kettle of fish and certain guidelines can be drawn from experience: viral proteases such as TEV, TVMV, etc. are precise and somewhat slow (typical use ratio is 1:10-1:50). Thrombin is very fast and furious (typical use ratio 1:1000 to 1:5000) but it can sometimes cause undesired cutting if you have either additional sites
[ccp4bb] Handling protein crystals from 20% isopropanol
Dear All: I have obtained good crystals from 20% isopropanol but this is my first time dealing with this agent. Help in handling the crystals, dealing with evaporation etc. is highly appreciated. Thank you Subbu
[ccp4bb] Baculovirus expression and purification - off topic
Dear All: I have a peculiar problem with baculovirus expression of my protein. The native protein elutes in Tris gradient. This has no problems. However, a mutant elutes in the wash. After further purification with size exclusion, I notice that this mutant is always associated with some medium component that I cannot separate. I know this (?) because when lyophllized (not a sin for this protein), I get 15-30 mg of protein (or colorless fluffy material) whereas protein estimation gives only about 5 mg. This amount is what I usually get for the native protein. I have tried active charcoal, BioRex, ammonium sulfate precipitation and even hydroxyapatite columns but nothing seems to work to separate the extra material from the protein. Any insight would be of much help. Thanks a lot Subbu
[ccp4bb] Activity of a mutant enzyme compared to wild type - puzzle
Dear all: I have a single residue mutant whose enzyme activity is about 50% of the wild type. Interestingly, the mutation is in a region that involves a secondary site but not the active site. The two structures with or without ligands fit well (0.18 A) and the metal binding and cofactor binding sites are all preserved in the mutant. The one difference noticed is that the ligand does not fill the active site (partially occupied subsites) unlike the wild type where all the subsites are occupied. Water structure around the actives site residues are identical. I looked at the electrostatics and both surfaces look similar (not an expert). There are some residues whose sides chains show some positional disorder and these residues are at the edges of the active site. The resolution of the both data sets are 1.5A. The mutant enzyme was derived by MR. One another possibility that I want to look at is to compare the compactness of the two enzyme structures. What is the best way to compare that? I am wondering whether the breathing that was mentioned for some enzymes might be playing a role in the mutant enzyme. Also, I would appreciate comments on other possible explanations for this unusual (?) behavior. Thanks a lot Subbu
[ccp4bb] Negative density around C of COO-
Dear all: I am noticing that in some of my structures, at 1.5 A resolution, 1) there is some negative density around the C of the carboxyl groups. 2) I also notice the negative density around S in a disulfide region. It is as though the disulfide is broken. Could some body enlighten me on this feature?
[ccp4bb] Summary: Negative density around COOH
Thanks for the quick reply and pointing out t bunch of references. Radiation damage has been pointed out as a cause for such feature. The following references were pointed out. Ravelli, R.B.G. McSweeney, S.M. (2000). The 'fingerprint' that X-rays can leave on structures. Structure 8:315-328. Wiek et al. (2000) PNAS 97, 623-628, and Leiros et al. (2001) Acta Cryst. D 57, 488-497. Subbu
[ccp4bb] How to generate atoms lying on plane - surface generation
Dear All: Is there a simple graphical way to generate atoms that lie on a surface (and wite out a pdb file) for modeling surface docking experiments? Thanks a lot Subbu I am looking for Cerius2 type but any code will be fine
[ccp4bb] Summary: Is phophorylation possible in E. coli expression system?
Dear All: Thanks for all your replies to this. Phosphorylation is possible in E. coli. One article of particular interest is Mol Cell Proteomics 2007 Eprint by Macek B, Gnad, F, Soufu B, Kumar C, Olsen JV, Mijakovic, I and Mann M. Many others have pointed out that it is possible and many likely residues such as Asp, Thr/Ser, His and Tyr are candidates for phosphorylation. To those who wanted to know whether the protein I am working on is a kinase: No, it is not. Thank you all again Subbu
[ccp4bb] Is phophorylation possible in E. coli expression system?
Dear All: This is not crystallography related and does not belong to this group, but I would like to pose this to all who are working with proteins expressed in an E. coli BL21 (DE3) or Rosetta. Is it possible for a protein to be phosphorylated during expression? At least my understanding was that post-translational modifications are not possible in E. coli. However, recently we expressed a protein and noticing that there are lot of potential tyrosine phosphorylation sites, we checked the expressed protein on a gel using a stain that detect phosphates. (Please do not say Are you crazy? Why would you check.. etc). Lo and behold, there lights up the band. Hence my question to you all. I could not google or mine from PubMed specific references that exist regarding this. Please enlighten me. Thanks a lot. Also, I would like to take this opportunity to thank everybody for all the help whenever ask some off-topic question like ths. Subbu
[ccp4bb] Predict potential sites for introducing cys : Summary
Dear All: Thanks for the overwhelming suggestions. I had in fact used SSBOND. However, there were other sugestions which are listed below. ssbond: http://eagle.mmid.med.ualberta.ca/forms/ssbond.html disulfide by design: http://www.ehscenter.org/dbd/ http://www.predictprotein.org/ - have not checked yet http://contact.ics.uci.edu/bridge.html - DIPro Some useful references were also provided Sowdhamini R, Srinivasan N, Shoichet B, Santi DV, Ramakrishnan C, Balaram P. Protein Eng. 1989 Nov;3(2):95-103 There is also a sequel to this paper in 2003. Thanks again Subbu
[ccp4bb] Predict potential sites for introducing cys to make a disulfide
Dear All: I vaguely remember a program that can predict sites for introducing a pair of Cys residues to make a disulfide. I had once used this before but now I had forgotten about it. Does any body know about this? My plan is introduce Cys residues into my structure that I can use for FRET analysis. Any suggestions would be greatly appreciated. Thanks Subbu
[ccp4bb] Dnase activity in E. coli expressed his tag proteins
Dear All: This is off topic but I would rather try here first. I am wondering whether Dnase could be a contaminant during the nickel affinity purification of a his.tag protein expressed using pET29b. The cells were disrupted using sonication only. Very high yield (30 mg/liter of cell culture). FPLC purification. SDS-PAGE shows a singe band even when overloaded. Silver staining is in progress. The protein I am interested in does not have any similarity to Dnase. However, when treated with supercoiled dna, the dna is degraded as efficiently as with Dnase I from Sigma. I am looking for ways to show that my preparation does not have any Dnase contamination. I would appreciate it very much if someone points me in the right direction. Thanks a lot Subbu
