Hi Hay, I think SAXS should be more than capable of discriminating between a 12.5 kDa monomer vs ~37.5 kDa trimer.
Lysozyme is a useful standard used in SAXS (as with most structural biology!), and Lysozyme is only slightly larger than your proteins. Cheers, Dave On Fri Dec 12 2014 at 5:13:26 PM Hay Dvir <hd...@tx.technion.ac.il> wrote: > Tanner: > Thanks, GREAT reference on asymmetric homo oligomers! > SAXS sounds like a good idea for a bit larger particles. I'm afraid it > might be very difficult to get enough resolution to resolve oligomerization > of a rather small 12.5 kDa protein like ours, but will look into it more > closely. > > Joes: > Thanks, we are aware of the serious problem of instability of asymmetric > homo-oligomers which could essentially polimerize as you nicely explain and > cite. Indeed one of the hypothesis we aim to test if we get additional > evidence about the the trimeric assembly concerns its known function to > interact with another protein, which could potentially provide the > complementary quaternary stability. Interface mutational analysis sounds > like a good approach to take in such cases. > > Thanks again an very best, > Hay > > > On Dec 12, 2014, at 5:39 PM, Tanner, John J. wrote: > > Two thoughts on asymmetric oligomers. > > 1. Here is a recent survey of asymmetric homodimers in the PDB. I know > you are looking for trimers, but at least this provides a precedent for > asymmetric oligomers. > > Swapna LS, Srikeerthana K, Srinivasan N. Extent of structural asymmetry > in > homodimeric proteins: prevalence and relevance. PLoS One. > 2012;7(5):e36688. doi: > 10.1371/journal.pone.0036688. Epub 2012 May 22. PubMed PMID: 22629324; > PubMed > Central PMCID: PMC3358323. > > 2. SAXS is a very effective method for determining whether assemblies > observed in crystals are stable in solution, since it provides not only the > oligomeric state, but also the quaternary structure. The oligomeric state > can be obtained from the volume of correlation (1) and Porod-Debye analysis > (2). The quaternary structure can be deduced by comparing the experimental > SAXS curve to theoretical curves calculated from oligomer models identified > by PISA or from manual inspection. The FoXS server and CRYSOL are good > tools for this. FoXS also allows ensembles of oligomers (MES) to be used in > fitting the data (e.g. mixture of monomer + dimer). I believe ATSAS also > has an ensemble program, but the name escapes me at this time. We have > used this approach to show that assemblies that are predicted to be stable > by PISA are not found in solution (3 and unpublished results). > > 1: Rambo RP, Tainer JA. Accurate assessment of mass, models and > resolution by > small-angle scattering. Nature. 2013 Apr 25;496(7446):477-81. doi: > 10.1038/nature12070. PubMed PMID: 23619693; PubMed Central PMCID: > PMC3714217. > > 2: Rambo RP, Tainer JA. Characterizing flexible and intrinsically > unstructured > biological macromolecules by SAS using the Porod-Debye law. Biopolymers. > 2011 > Aug;95(8):559-71. doi: 10.1002/bip.21638. Epub 2011 Apr 20. PubMed PMID: > 21509745; PubMed Central PMCID: PMC3103662. > > 3: Luo M, Singh RK, Tanner JJ. Structural determinants of > oligomerization of > δ(1)-pyrroline-5-carboxylate dehydrogenase: identification of a > hexamerization > hot spot. J Mol Biol. 2013 Sep 9;425(17):3106-20. doi: > 10.1016/j.jmb.2013.05.027. > Epub 2013 Jun 7. PubMed PMID: 23747974; PubMed Central PMCID: PMC3743950. > > On Dec 12, 2014, at 4:56 AM, Jose Manuel Duarte wrote: > > Dear Hay > > Your post prompted me to respond, since I think the issue of symmetry is > extremely important. > > I would like to reinstate here what should be obvious to everyone: a > stable asymmetric assembly of proteins in solution is essentially > impossible (or at most very very unlikely), purely because of topological > reasons. > > This is beautifully explained in a classic paper now 50 years old: Monod, > Wyman, Changeux (1965) "On the Nature of Allosteric Transitions: A > Plausible Model". The reasoning there is that a homomeric protein in > solution can only associate in 2 ways: isologous (binding with same surface > patches in both monomers, necessarily through a 2-fold axis) or > heterologous (binding through different surface patches in both monomers). > The isologous case is clearly symmetric (C2). Whilst in the heterologous > case the monomers can either assemble infinitely or form a closed > symmetry. The conclusion that follows is that stable homo-oligomers can > only be symmetric. > > I especially like this paragraph: > > "On the basis of these considerations, it is reasonable to assume that, if > an oligomeric protein possesses a wide range of stability, it consists of a > closed structure where all the protomers use the same binding sets; which > implies, as we have just seen, that the molecule should possess at least > one axis of symmetry." > > The paper really explains it a lot better than me, it can be found here: > http://www.pasteur.fr/ip/resource/filecenter/document/01s-00004j-0er/monod-wyman-changeux-1965.pdf > > The conclusion in any case is that asymmetry in homomers is, if not > impossible, highly unlikely. So in my opinion asymmetric assemblies should > be proposed with a lot of care, only if experimental data really is > overwhelmingly clear. For instance I don't think that gel filtration or AUC > would be good evidence enough: it really needs to be demonstrated that the > interface that you see in the crystal is the one leading to oligomerisation > (perhaps with a mutagenesis experiment?). Otherwise the interface in the > crystal is most likely simply a crystal contact. > > Jose > > > > On 12/12/14 10:15, Hay Dvir wrote: > > Dear Jeremy, > > Indeed, we also incline to think of it as a monomer in solution, but > still quite un-eased by the extensive interactions in the asu being merely > as a result of a crystallization artifact. As you said, we may need to rely > more heavily on biochemical analysis and since SEC wasn't clear we are > turning now to LS (hope to able to post a more conclusive update). > > Regardless of what our final conclusion would be for this case, we > became rather generally interested to find other similar cases of > *homomeric* assemblies related only by non crystallographic translation > symmetry (or as Engin Qzka pointed out "improper NCS" is the conventional > terminology). So to rephrase our question we are interested to learn about > additional structures of *homomoeric improper ncs assemblies*. > > I truly appreciate ANY open-minded or skeptic thought, profound or > trivial that we get here! They all, definitely those made by Mark Garavito, > contribute to shaping our mind around this riddle. > Thanks for commenting on the skepticism, I brought it up as part of the > discussion but a glitch of my own coffee time haziness might have slipped > in. Perhaps I should try some o-cha instead .. :) > > cheers, > Hay > > > On Dec 12, 2014, at 3:05 AM, Jeremy Tame wrote: > > Dear Hay > > I suggest that you use analytical ultracentrifugation to determine the > oligomeric state of the protein in solution. > Mass spectrometry and light scattering are also useful, but there are so > many examples of gel filtration proving > erroneous it has questionable value as an analytical technique. For an > example of a dimer interface predicted by > PISA to be real you could look at Yoshida et al, JMB 423, 351 (2012). The > protein is in fact a monomer in solution. > PISA is a fantastic tool, but interfaces in crystals do not always reflect > the solution state. My guess (with the > information I have) is that your protein is probably a monomer too. > > With regard to Michael Garavito's reply requesting more information, I > would like to comment that scepticism > is indeed an important god in the pantheon of science, but that that minor > deity open-mindedness also deserves the > occasional nod. 10-fold crystal symmetry is one example, but the list of > "impossible" things now become mainstream > is a long one (continental drift, Earth >100,000 years old, quantum > mechanics....and so on). Bayes theorem cannot > help you discover the truth if you have set its prior probability to zero. > But I haven't my morning o-cha yet either. > > good luck > Jeremy > > > On Dec 11, 2014, at 9:27 PM, Hay Dvir wrote: > > Dear all, > > > > We have a structure of a rather tightly packed homotrimer protein in the > ASU with no apparent crystallographic or non-crystallographic rotational > symmetry between monomers. > > Attempting to establish the biological assembly, we are very interested to > hear about additional similar cases you might know of. > > > Thanks in advance, > > Hay > > > > --------------------------- > > Hay Dvir Ph. D. > > Head Technion Center for Structural Biology > > Technion Haifa 3200003, Israel > > Tel: +(972)-77-887-1901 > > Fax: +(972)-77-887-1935 > > E-mail hd...@technion.ac.il > > Website http://tcsb.technion.ac.il > > > > > > > >
