Re: [ccp4bb] Protein melting temperatures
Dear Raji, what exactly do you mean when you say the melting temperature is 45deg. Did you only test one buffer, or did you test many buffers and 45deg is the most stable one? If you have only tested one buffer you should run a screen testing different buffer systems (pH) and e.g. NaCl concentration and glycerol concentrations (or ligands, if your proteins binds any). Then you identify the buffer which is stabilizing your protein the most. I have seen big impacts on protein stability and crystallization when optimizing my buffers like this. I think you should not only consider the melting temperature alone, but also how the curve looks like. Do you get a high initial flourescence (which often indicates partially unfolded protein or hydrophobic patches) or do you have very low initial flourescence (which is a good sign for compact protein). Another thing to look at is if your transition is sharp (the steeper the better). Taking all this together you can judge if your protein is happy or not. Hope this helps you! Linda Patrick Shaw Stewart wrote: I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark
Re: [ccp4bb] Apparent twinning in P 1 21 1
Dear Yuri, in a monoclinic space group an orthorhombic lattice metric can be simulated when one of the following conditions is fulfilled: i) a = c [e.g. in Wittmann Rudolph (2007) Acta Cryst. D63, 744-749], ii) the beta angle is close to 90° [e.g. in Larsen et al. (2002) Acta Cryst. D58, 2055-2059 ] or iii) c cos beta is about -a/2 [e.g. in Declercq Evrard, (2002) Acta Cryst. D57, 1829-1835]. The a and b axes of the orthorhombic cell are identical to the monoclinic a and c axes, respectively. The length of the orthorhombic b-axis can also be calculated by c(monoclinic) cos(beta-90°) = 1/2b(orthorhomic). I would assume that you have the case iii with a quite high twin fraction. If I recall correctly, Declercq and Evrard have a nice figure in their paper showing the geometric relationship. If not, let me know and I can sent you a figure. Good luck! Linda Yuri Pompeu schrieb: Hello everyone, I have a 2.3A data set that could be scaled in C 2 2 21 and P 1 21 1 Intensity statistics tests indicate twinning (pseudo-merohedral h,-k,-h-l in P 1 21 1) I find a good MR solution and when I try to refine it with the twin law I get fairly good maps and decent Rs 21-28%. I can see features tha were not in the search model Which leads me to think that this a valid solution. The one thing that bothers me however is the fact that my beta angle in P 1 21 1 is 104 (not close to 90) and that the geometry gets worse after refinement? Any suggestions? cheers *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark
Re: [ccp4bb] Low resolution refinement
Dear Joane, we had a case, where we had five molecules in the assymmetric unit where the biological functional unit was a homotrimer. So we had one non-crystallographic trimer and two monomers, which were located along the 3-fold symmetry axis of space group I213. One of the monomers also showed electron density of considerably lower quality, obviously going along with higher B-factors. By a careful analysis of the crystal packing we could see that this chain has only very few crystal contacts. If you want to have a closer look, see: Schuldt L, Weyand S, Kefala G, Weiss MS J. Mol. Biol. (2009), 863-879. The Section Crystal Packing and structural variation describes this in more detail. Best wishes, Linda Joane Kathelen Rustiguel schrieb: Dear all I am refining a structure at 3.4 A resolution that contains 3 molecules in the a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric functional structure expected for this class of proteins. The other two chains B and C, which also form the functional dimer, seem to be, somehow, a lot more flexible than chain A. As a result, whereas the electron density map, b-factor and geometry for chain A is pretty reasonable for a 3.4 A resolution structure, the refinement for the other two chains (B and C) does not behave well. Even playing with different weights for geometry, analysing different levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map for the helical regions is ok, but the electron density map for strands and loops of chains B and C are broken along the main chain, B-factors are really high, and the geometry keeps being distorted. Right now, the R-factor and R-free are 24.2 and 28.6, respectively. Any suggestions in how to proceed the refinement? And even a more difficult question, how do we report this type of structure? How do we deposit those coordinates? We can certainly use chain A as a model to perform interesting studies of structure-function relationship, but we know that chain B and chain C have problems. Any help will be greatly appreciated. Regards Joane -- Joane Kathelen Rustiguel Bonalumi Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP Laboratório de Cristalografia de Proteínas Departamento de Física e Química Fone: +55.16.3602.4193 *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark
Re: [ccp4bb] DTU vs DTT ?
Hi Emmanuel, it is hard for me to imagine that Coot has the wrong stereoisomer. So what I think might have happend is the following: You have imported the correct DTT, but when you have fitted the molecule into the map you might have distorted the sterochemistry at the C3 atom. And then it was refined like that. I had once a similar observation with MPD. Did you insert the three DTT molecules individually, or did you copy and paste the same molecule around (which might explain why you have it for all three)? And why don´t you just import DTT into Coot and check yourself if it has the correct sterochemistry. This is a fast and easy way to find out if something is wrong in your Coot library. Unless you have very high resolution which clearly shows that you have DTU instead of DTT, I would change your coordinate file to DTT and send it again to the pdb. Hope this helps you. Best wishes, Linda Emmanuel Saridakis schrieb: Dear All, Possibly a trivial question but your experience would be much appreciated: I recently submitted a structure to PDB containing 3 DTT (dithiothreitol) molecules, or so I thought. The molecules had been imported and fitted with Coot using the Get Monomer... instruction with the code DTT. The Annotator responded, quite rightly as it turns out, as follows: Please note DTT in your coordinates has been changed to DTU since it has incorrect stereochemistry as DTT. Please review the stereochemistory in the attached validation report summary. You can send me corrected stereochemistry for DTT if you want it changed back. DTU (2R,3S)-1,4-disulfanylbutane-2,3-diol C4 H10 O2 S2 DTT (2R,3R)-1,4-disulfanylbutane-2,3-diol C4 H10 O2 S2 So, is the DTT monomer of Coot in fact its stereoisomer known as dithioerythritol? Should I import the correct DTT from elsewhere and re-refine or is there something else behind this? Thanks a lot for any suggestions! Emmanuel
Re: [ccp4bb] Strange density on Serine oxygen.
Hi Vinson, along these lines: did you check the molecular weight of your protein with MS? This should help to answer if the molecular weight deviates from the expected one. Best wishes, Linda Savvas Savvides schrieb: Hi Vinson Beyond the possibility for another type of residue as already suggested by Phil and Mark, there is also the possibility of O-linked glycosylation of the serine and threonine, if your protein undergoes such post-translational modification and it has been expressed via an expression system that processes the protein in that way. Ser/Thr tandems are well known targets for O-glycosylation (http://www.cbs.dtu.dk/databases/OGLYCBASE/). best regards Savvas Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Ph. +32 (0)472 928 519 http://www.LProBE.ugent.be/xray.html On 24 Nov 2010, at 13:10, Vinson LIANG wrote: Dear all, I'm refining a structure and find some strange triangle density on the oxygen of Ser and Thr at the C terminus. One picture of the strange density is attached here. Could anyone please give me some suggestions on what this could be? The buffer used during purification is PBS, Tris and NaCl. And crystallization condition contains PEG3,350 and Mg(NO3)2. Thank you all in advance for any suggestion. Best, Vinson Liang triangle_density.gif
