Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
The problem was resolved by Isabel Uson and Giovanna Petrillo using ARCIMBOLDO! The real spacegroup is P21, twinned to look like C2221. I would probably take years to solve that on my own, they did it very fast. This was a difficult problem, and still is, since if I try MR using the correct solution and space group I cannot resolve it. I definitely recommend everybody with difficulties to give Arcimboldo a try. Thank you Isabel! Thank you Sergei Strelkov, your message was very informative as usual. ;) Thank you George M. Sheldrick for the information on how to discrimitate between correct and wrong MR solutions using SHELXE. Best regards, Napo On 11/4/2011 7:42 AM, Sergei Strelkov wrote: Dear Napoleão, Thank you for updating everyone on your efforts, and also acknowledging the advice. I wanted to respond to your question regarding maps. I know that many people who try to figure out whether or not their MR solution is the right one would ask the same question. So first of all if you wonder why you actually get very decently looking maps the answer is a classical one: because 'the phases are more important than amplitudes'. The appearance of your map is defined by your model phases, and hence a good match between the model and the map /may not/ be taken as a sign of a correct solution. Once again: /never ever!/ On the contrary, at least in your coil1.jpg image I clearly see that the density exactly follows the model which is almost entirely poly-Ala. Unless your protein is really poly-Ala this should be alarming. If you had a correct solution they you would hope to see the (difference)density for at least some missing side chains. And a second point. Unless your model contains the complete chain (which is rarely the case, especially for the coiled coils, as discussed already) a sign of the correct solution would be the appearance of extra density near the N- and/or C-terminus of the model. If it is not there, it is almost certainly not a solution. And you should not be worried about the R-factors being very high at this stage. If the solution is correct then you should see at least some extra features in the map. Kind regards, Sergei Thank you all for the replies. Sorry for taking so long to reply, I was actually trying some of your interesting ideas (and I'm still trying). I tried using the low resolution data sets for the molecular replacement (thanks to Yuriy Patskovsky), I also improved and increased my coiled coil database and employed it in many approaches using EPRM (interesting program I was not aware of), which I found to produce lots of data, hopefully addressing at some extent the helixes bent (thanks to Bernhard Rupp). I also tried some more tweaking in Phaser, although not sure if did it properly (thanks to Randy Read). There is no twinning as far as I can tell (thanks to Ed Pozharski for the tip). Using a data set with enough completeness (360 degrees @ Brookhaven) and processing in P1 did not help me because in this space group there is most likely 2-3 helixes in the asymmetric unit, which complicates the problem (and it takes a lot of time for Phaser to run). Automated approaches also did not yield a better result (as far as I can tell). I'm convinced that the space group is C2221, but I may be wrong. Thanks to Sergei Strelkov for the numerous useful suggestions on how to approach the problem. One of the big issues for me is to discriminate between a lot of similarly good density maps. For example: http://www.fullonline.org/coils/coil1.jpg http://www.fullonline.org/coils/coil2.jpg I have hundreds of solutions like these and I think they are all wrong. I couldn't manage to run Arcimboldo, could not find a tutorial on it either. It was highly recommended here (and elsewhere), so I'm definitely willing to give it a try (thanks Isabel Uson). You guys opened my eyes about a series of issues that I should learn about and approach, I'm most thankful for that. Best regards, Napo -- Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven O&N2, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium Work phone: +32 16 330845 Fax: +32 16 323469 OR +32 16 323460 Mobile: +32 486 294132 Lab pages:http://pharm.kuleuven.be/anafar
[ccp4bb] Merging with CAD fails
Hello, I am trying to merge two data sets on from 28 to 2.3 A 99% comlpeteness with another one from 26 to 1.95A 82% completeness. I keep getting an error saying Duplicate labels in the output file. I am sure its something simple but I cannot seem to figure it out Any ideas? Thanks
Re: [ccp4bb] Merging with CAD fails
If you post the cad input file, it should be easy to pinpoint the problem. As it stands, you are either: 1) Including Miller indices as merged columns - they get done automatically, so if you specify them, you get the duplicate labels 2) You actually do have the same name for the two columns in the output - thus duplicate labels 3) You may be thinking that cad can merge two datasets into one - it can't. CAD just takes columns from two or more files and puts them into one. What you are trying to do requires scaling/merging of two datasets - you should look at scala for that. HTH, Ed. -- Hurry up, before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Merging with CAD fails
Thanks for the help. I believe option 3 describes my situation the best. I am looking into it now... Best, Yuri On Sat, 05 Nov 2011 16:38:11 -0400, Ed Pozharski wrote: If you post the cad input file, it should be easy to pinpoint the problem. As it stands, you are either: 1) Including Miller indices as merged columns - they get done automatically, so if you specify them, you get the duplicate labels 2) You actually do have the same name for the two columns in the output - thus duplicate labels 3) You may be thinking that cad can merge two datasets into one - it can't. CAD just takes columns from two or more files and puts them into one. What you are trying to do requires scaling/merging of two datasets - you should look at scala for that. HTH, Ed. -- Yuri Pompeu
Re: [ccp4bb] Merging with CAD fails
Hi You can use Pointless to merge the files together into one mtz file - then take the output mtz from Pointless and run it through Scala (though its replacement program Aimless is faster and seems to do a better job, and is available directly from Phil Evans' ftp site). I think the original problem arises from having reflection columns with the same label in each of the two original mtz files - I think Pointless takes care of this for you (but it is a Saturday night...). On 5 Nov 2011, at 20:41, Yuri wrote: Thanks for the help. I believe option 3 describes my situation the best. I am looking into it now... Best, Yuri On Sat, 05 Nov 2011 16:38:11 -0400, Ed Pozharski wrote: If you post the cad input file, it should be easy to pinpoint the problem. As it stands, you are either: 1) Including Miller indices as merged columns - they get done automatically, so if you specify them, you get the duplicate labels 2) You actually do have the same name for the two columns in the output - thus duplicate labels 3) You may be thinking that cad can merge two datasets into one - it can't. CAD just takes columns from two or more files and puts them into one. What you are trying to do requires scaling/merging of two datasets - you should look at scala for that. HTH, Ed. -- Yuri Pompeu Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
[ccp4bb] yellow crystals
Hi -- has anyone had crystals that are colored in regular (unpolarized) light? Mine are yellow, and I'm not aware of anything in the buffer conditions that might cause this. I read online that glutaraldehyde can turn protein crystals a golden color, but as far as I know there isn't any of that in the well. Purified in HBS pH 7.2; crystallized in LiCl/PEG4K/Tris pH 8. Any explanations? Thanks! Caitlyn Caitlyn C. Yeykal Mrksich Group/Adams Group Dept. of Biochemistry, University of Chicago 929 E. 57th St., Rm 547B Chicago, IL 60615 cait...@uchicago.edu
Re: [ccp4bb] yellow crystals
On Sat, Nov 5, 2011 at 3:02 PM, Caitlyn Claire Yeykal wrote: > Hi -- has anyone had crystals that are colored in regular (unpolarized) > light? Mine are yellow, and I'm not aware of anything in the buffer > conditions that might cause this. I read online that glutaraldehyde can > turn protein crystals a golden color, but as far as I know there isn't any > of that in the well. Purified in HBS pH 7.2; crystallized in > LiCl/PEG4K/Tris pH 8. Any explanations? Many cofactors can cause this - the ones I'm familiar with are LLP and NAD, but there are quite a few others, and they will often co-purify with the proteins that bind them. Possibly certain metals as well, but I'm less certain about that. You may be able to identify the responsible molecule from the absorbance across the visible light spectrum, although I found it difficult to track down information on peak absorbances when I last tried this several years ago. -Nat
Re: [ccp4bb] yellow crystals
Thanks for all the replies -- there are no suggestions in the literature or in crystallized or predicted domain structures that this protein binds a cofactor, and, although I did purify it in insect cells, PAGE gels and activity assays support the assertion that it's not ferritin. Nobody has seen any metal ions bound, either, but there are a few domains that haven't been crystallized, so maybe. Again, thanks for all the possibilities; will keep them in mind. Caitlyn On Nov 5, 2011, at 5:02 PM, Caitlyn Claire Yeykal wrote: Hi -- has anyone had crystals that are colored in regular (unpolarized) light? Mine are yellow, and I'm not aware of anything in the buffer conditions that might cause this. I read online that glutaraldehyde can turn protein crystals a golden color, but as far as I know there isn't any of that in the well. Purified in HBS pH 7.2; crystallized in LiCl/PEG4K/Tris pH 8. Any explanations? Thanks! Caitlyn Caitlyn C. Yeykal Mrksich Group/Adams Group Dept. of Biochemistry, University of Chicago 929 E. 57th St., Rm 547B Chicago, IL 60615 cait...@uchicago.edu
Re: [ccp4bb] yellow crystals
It's also possible that there's oxidation in the buffer causing the yellow color. I'm not sure how common this is with HEPES. But I see it all the time with MOPS. Alternatively, does the yellow color bind the column during purification? If so, then it sounds like a co-purified flavin or protein. Best regards, Reginald McNulty On Nov 5, 2011, at 5:57 PM, Caitlyn Claire Yeykal wrote: > Thanks for all the replies -- there are no suggestions in the literature or > in crystallized or predicted domain structures that this protein binds a > cofactor, and, although I did purify it in insect cells, PAGE gels and > activity assays support the assertion that it's not ferritin. Nobody has > seen any metal ions bound, either, but there are a few domains that haven't > been crystallized, so maybe. Again, thanks for all the possibilities; will > keep them in mind. > > Caitlyn > > On Nov 5, 2011, at 5:02 PM, Caitlyn Claire Yeykal wrote: > >> Hi -- has anyone had crystals that are colored in regular (unpolarized) >> light? Mine are yellow, and I'm not aware of anything in the buffer >> conditions that might cause this. I read online that glutaraldehyde can >> turn protein crystals a golden color, but as far as I know there isn't any >> of that in the well. Purified in HBS pH 7.2; crystallized in >> LiCl/PEG4K/Tris pH 8. Any explanations? >> >> Thanks! >> Caitlyn >> >> >> Caitlyn C. Yeykal >> Mrksich Group/Adams Group >> Dept. of Biochemistry, University of Chicago >> 929 E. 57th St., Rm 547B >> Chicago, IL 60615 >> cait...@uchicago.edu >
Re: [ccp4bb] yellow crystals
In another thread, you indicated that there were no identifiable cofactor binding sites in your protein, so we are down to less common situations. Some proteins are spontaneously decorated with pyridoxal on surface lysine residues. In some cases, this has absolutely nothing to do with the enzymatic activity of the protein. On Nov 5, 2011, at 5:02 PM, Caitlyn Claire Yeykal wrote: > Hi -- has anyone had crystals that are colored in regular (unpolarized) > light? Mine are yellow, and I'm not aware of anything in the buffer > conditions that might cause this. I read online that glutaraldehyde can turn > protein crystals a golden color, but as far as I know there isn't any of that > in the well. Purified in HBS pH 7.2; crystallized in LiCl/PEG4K/Tris pH 8. > Any explanations? > > Thanks! > Caitlyn > > > Caitlyn C. Yeykal > Mrksich Group/Adams Group > Dept. of Biochemistry, University of Chicago > 929 E. 57th St., Rm 547B > Chicago, IL 60615 > cait...@uchicago.edu
Re: [ccp4bb] Merging with CAD files
Hi, I used CAD for merging datasets during MIR. I faced the same problem. The solution is: the datasets you are trying to merge should have different labels i.e if dataset 1 has labels: F and SigF, dataset 2 should be F_d1 and SigF_d1. Mention the labels during the cad run. Ramanuj Banerjee Crystallography and molecular Biology Division Saha Institute of Nuclear Physics, Kolkata, INDIA
