Re: [ccp4bb] asymmetric homotrimer in the asu
Hay, Indeed, we also incline to think of it as a monomer in solution, It is in fact possible that different solution conditions favor different oligomeric assemblies. For example, perhaps your protein, which in one set of solution conditions prefers the monomer, prefers some other assembly under the crystallization conditions (we've seen this in our own work). Perhaps the higher (or lower) salt concentration, the presence (or absence) of the precipitation reagent, etc. pushes any oligomer equilibrium that exists for your protein. 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*. An interesting case of heterologous interfaces engaged is that of the VP40 protein. A recent publication from the Saphire group (PMID 23953110 http://www.ncbi.nlm.nih.gov/pubmed/23953110) may present an instructive situation, though I don't know the details of the NCS observed. Here an octameric assembly (PDB 4LDM, ASU=monomer) and a hexameric assembly (PDB 4LDD, ASU=dimer) of the same protein engage different interfaces on the protomers. In forming the oligomer, the protomers are aligned with alternating interfaces. Emily. 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 mechanicsand 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 323, Israel Tel: +(972)-77-887-1901 Fax: +(972)-77-887-1935 E-mail hd...@technion.ac.il Website http://tcsb.technion.ac.il
Re: [ccp4bb] asymmetric homotrimer in the asu
It is in fact possible that different solution conditions favor different oligomeric assemblies. For example, perhaps your protein, which in one set of solution conditions prefers the monomer, prefers some other assembly under the crystallization conditions (we've seen this in our own work). Perhaps the higher (or lower) salt concentration, the presence (or absence) of the precipitation reagent, etc. pushes any oligomer equilibrium that exists for your protein. - this all is very true; in addition, oligomeric state does depend on protein concentration, too. jsPISA at CCP4 (http://www.ccp4.ac.uk/pisa) calculates concentration profiles for probable macromolecular assemblies, which may be useful to check with. The effect of salt/precipitators/pH is not there, though. Eugene 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. An interesting case of heterologous interfaces engaged is that of the VP40 protein. A recent publication from the Saphire group (PMID 23953110http://www.ncbi.nlm.nih.gov/pubmed/23953110) may present an instructive situation, though I don't know the details of the NCS observed. Here an octameric assembly (PDB 4LDM, ASU=monomer) and a hexameric assembly (PDB 4LDD, ASU=dimer) of the same protein engage different interfaces on the protomers. In forming the oligomer, the protomers are aligned with alternating interfaces. Emily. 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 mechanicsand 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 323, Israel Tel: +(972)-77-887-1901tel:%2B%28972%29-77-887-1901 Fax: +(972)-77-887-1935tel:%2B%28972%29-77-887-1935 E-mail hd...@technion.ac.ilmailto:hd...@technion.ac.il Website http://tcsb.technion.ac.ilhttp://tcsb.technion.ac.il/
Re: [ccp4bb] asymmetric homotrimer in the asu
On 12.12.2014 23:53, jt...@tsurumi.yokohama-cu.ac.jp wrote: When you state however that it is highly unlikely for a homo-oligomer to show asymmetry, I think you are forgetting the well-known phenomenon of half-of-sites reactivity among enzymes. A simple internet search will in fact show many examples where two copies of the same molecule form a complex, adopting different conformations to do so. Symmetry-breaking is a general feature of Nature, so it is not too surprising that proteins may adapt to a partner's presence in this way. One has to be careful with what we mean with the word asymmetry. Monod's reasoning simply says that a single molecule can't associate in a heterologous way unless the open binding surfaces are closed by symmetry. Thus it precludes asymmetry in the sense of the quaternary structure assembly of homomers, it does not say however that the subunits of a homomer can't have slight conformational differences. The quaternary structure is still symmetric (not in the strict crystallographic sense of perfect mathematical symmetry, but in the sense of approximate symmetry resulting in superpositions with low rmsds). The most important concept here is that the binding interfaces MUST be symmetric due to topological reasons: either in the isologous sense (2-fold symmetry, face-to-face binding) or in the heterologous sense (cyclic symmetry where the molecules associate in a face-to-back fashion, where both the face and back sites are satisfied simultaneously in every molecule in the assembly). Levy and Teichmann have a very nice review on this: http://www.sciencedirect.com/science/article/pii/B978012386931927 In any case the half-of-sites reactivity phenomenon is of course perfectly possible, there's nothing in the MWC paper against that idea. The main point I was trying to make is: if a crystal is composed of only one type of protein molecule (no ligands or extra molecules around) one should not conclude that it assembles in an asymmetric way through open heterologous interfaces. If there's no other molecules around to produce the asymmetry, then what you see in the crystal is a bunch of crystal contacts. Jose
Re: [ccp4bb] asymmetric homotrimer in the asu
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-4j-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 mechanicsand 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
Re: [ccp4bb] asymmetric homotrimer in the asu
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-4j-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
Re: [ccp4bb] asymmetric homotrimer in the asu
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-4j-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
Re: [ccp4bb] asymmetric homotrimer in the asu
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:
Re: [ccp4bb] asymmetric homotrimer in the asu
Hi everyone, Just wanted to second the SAXS suggestion: the approach should provide a very rigorous way of testing the candidate models apparent in the crystallographic lattice, and can assist with questions of mass. (among the most common applications) With regards to the mass question, test as many concentrations as you can to get a pulse on the strength of the interaction, and also use other complementary solution methods such as AUC or SEC-MALS. Note that the concentrations tested in SAXS are several fold higher than those examined in AUC or SEC/SEC-MALS, and may not be physiologically relevant in that regard. Indeed, a 12.5 kD monomer will have very weak scattering power (intensity varies as the square of molecular volume). You can compensate for that by increasing the concentration or using synchrotron radiation. The ‘rule of 100’ is a good guide – in your SAXS expt, start at a sample concentration where MW (kD) * concentration (mg/mL) ~100 and then adjust accordingly. (I’ve successfully studied molecules as small as a 8.5 kD Tudor domain and a ~7kD CHAPS micelle in this way.) Hope that helps, Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Group Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania mailto:kgu...@upenn.edu kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / http://www.stwing.upenn.edu/~kgupta www.stwing.upenn.edu/~kgupta From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David Briggs Sent: Friday, December 12, 2014 12:37 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] asymmetric homotrimer in the asu 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
Re: [ccp4bb] asymmetric homotrimer in the asu
Dear Jose Your email expressed far more clearly than mine the problem of symmetry, and I had completely forgotten this discussion in the classic MWC paper. The point about measuring the molecular weight in solution is to find IF the protein forms an oligomer or not - AUC can give some idea of shape (cigar or hamburger) but cannot give any further indication of asymmetry as SAXS can. However, if you find a monomer in solution, Hay's problem is solved. When you state however that it is highly unlikely for a homo-oligomer to show asymmetry, I think you are forgetting the well-known phenomenon of half-of-sites reactivity among enzymes. A simple internet search will in fact show many examples where two copies of the same molecule form a complex, adopting different conformations to do so. Symmetry-breaking is a general feature of Nature, so it is not too surprising that proteins may adapt to a partner's presence in this way. The bottom line is that MWC assumes symmetry, and this assumption is not always valid. Like Hay I would like to know of higher-order examples than dimer, as my group has some interesting symmetry-breaking results coming out... best wishes Jeremy ** 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. ...
[ccp4bb] asymmetric homotrimer in the asu
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 DvirPh. D. HeadTechnion Center for Structural Biology TechnionHaifa 323, Israel Tel:+(972)-77-887-1901 Fax:+(972)-77-887-1935 E-mail hd...@technion.ac.il Website http://tcsb.technion.ac.il
Re: [ccp4bb] asymmetric homotrimer in the asu
Dear Hay, And your point is? I am not trying to be snarky (although I am just starting my morning coffee), but to bring up the fact that CCP4BB readers need more info to comment on your case, like space group, local interactions, and how packed is tightly packed. I have had two cases of trimers, as my students initially called them, that were actually a dimer and a half. The half dimer had its mate in another ASU. Can it be a biological monomer that just happened to crystallize 3 monomers to an ASU? Non-symmetric homo-oligomers are rare, but sadly cannot be absolutely confirmed by crystallography alone, but by good old biochemistry. The PISA website (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) can give you estimations of the strengths of the interfacial interactions, but they are mere estimates. What does gel filtration say or cross linking? Does it fit with the biology/biochemistry expected of this protein? Anyway, have fun with your structure, but use a lot of skepticism in your interpretation. That will help you convince the reviewers. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Dec 11, 2014, at 7:27 AM, Hay Dvir hd...@tx.technion.ac.il 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 323, Israel Tel: +(972)-77-887-1901 Fax: +(972)-77-887-1935 E-mailhd...@technion.ac.il Website http://tcsb.technion.ac.il
Re: [ccp4bb] asymmetric homotrimer in the asu
bacterioferritin in the I422 space group, like 1JGC, has a trimer in the asymmetric unit, but the biological unit is 24-mer! Looking at it another way, considering a dimer is two monomers crosslinked by a heme, the asu contains 1.5 dimers and hence 1.5 hemes! eab On 12/11/2014 07:27 AM, 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 DvirPh. D. HeadTechnion Center for Structural Biology TechnionHaifa 323, Israel Tel:+(972)-77-887-1901 Fax:+(972)-77-887-1935 e-mailhd...@technion.ac.il mailto:hd...@technion.ac.il Websitehttp://tcsb.technion.ac.il
Re: [ccp4bb] asymmetric homotrimer in the asu
Dear Michael, Thank you very much for the useful comments. Indeed, we are of course looking at it biochemically, which isn't a clear cut so far.. As you pointed out it could be a monomer in solution, but the interface between monomers within this asymmetric trimer seems too extensive (compared to those responsible for the lattice packing) not to suspect a trimer as a solution assembly. PISA suggested this asymmetric trimer as the most likely assembly but it falls into the grey region of their criteria (see attached pic.) Since it's rare, we are interested to know of other similar reports, if any, to learn how they were resolved/concluded. I believe the case you describe is not similar, as we have NO rotational symmetry (2, 3, or 4-fold) whatsoever between interacting monomers in the ASU or relating those built up by the crystallographic symmetry. Therefore I can't see how the space group information may help, but it is p212121 in case it helps boosting your morning coffee experience with symmetry pondering ... :). Cheers, Hay On Dec 11, 2014, at 3:47 PM, R. M. Garavito wrote: Dear Hay, And your point is? I am not trying to be snarky (although I am just starting my morning coffee), but to bring up the fact that CCP4BB readers need more info to comment on your case, like space group, local interactions, and how packed is tightly packed. I have had two cases of trimers, as my students initially called them, that were actually a dimer and a half. The half dimer had its mate in another ASU. Can it be a biological monomer that just happened to crystallize 3 monomers to an ASU? Non-symmetric homo-oligomers are rare, but sadly cannot be absolutely confirmed by crystallography alone, but by good old biochemistry. The PISA website (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) can give you estimations of the strengths of the interfacial interactions, but they are mere estimates. What does gel filtration say or cross linking? Does it fit with the biology/biochemistry expected of this protein? Anyway, have fun with your structure, but use a lot of skepticism in your interpretation. That will help you convince the reviewers. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Dec 11, 2014, at 7:27 AM, Hay Dvir hd...@tx.technion.ac.il 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 323, Israel Tel: +(972)-77-887-1901 Fax: +(972)-77-887-1935 E-mail hd...@technion.ac.il Website http://tcsb.technion.ac.il
Re: [ccp4bb] asymmetric homotrimer in the asu
This fact by itself is unusual to say the least (for me): we have NO rotational symmetry (2, 3, or 4-fold) whatsoever between interacting monomers in the ASU or relating those built up by the crystallographic symmetry There might be several ways of choosing molecules to represent the asymmetric unit. Is it possible to find ones that are related? Say something like non-crystallographic translation (or pseudo translation) + non-crystallographic rotation. For long time I was thinking about such a possibility of having more than one molecule in au but no rotation or pst. May be I've missed but never found an evidence, nor can I explain why would that be impossible. Vaheh Oganesyan www.medimmune.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hay Dvir Sent: Thursday, December 11, 2014 12:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] asymmetric homotrimer in the asu Dear Michael, Thank you very much for the useful comments. Indeed, we are of course looking at it biochemically, which isn't a clear cut so far.. As you pointed out it could be a monomer in solution, but the interface between monomers within this asymmetric trimer seems too extensive (compared to those responsible for the lattice packing) not to suspect a trimer as a solution assembly. PISA suggested this asymmetric trimer as the most likely assembly but it falls into the grey region of their criteria (see attached pic.) [cid:image001.png@01D01549.A63DFAA0] Since it's rare, we are interested to know of other similar reports, if any, to learn how they were resolved/concluded. I believe the case you describe is not similar, as we have NO rotational symmetry (2, 3, or 4-fold) whatsoever between interacting monomers in the ASU or relating those built up by the crystallographic symmetry. Therefore I can't see how the space group information may help, but it is p212121 in case it helps boosting your morning coffee experience with symmetry pondering ... :). Cheers, Hay On Dec 11, 2014, at 3:47 PM, R. M. Garavito wrote: Dear Hay, And your point is? I am not trying to be snarky (although I am just starting my morning coffee), but to bring up the fact that CCP4BB readers need more info to comment on your case, like space group, local interactions, and how packed is tightly packed. I have had two cases of trimers, as my students initially called them, that were actually a dimer and a half. The half dimer had its mate in another ASU. Can it be a biological monomer that just happened to crystallize 3 monomers to an ASU? Non-symmetric homo-oligomers are rare, but sadly cannot be absolutely confirmed by crystallography alone, but by good old biochemistry. The PISA website (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) can give you estimations of the strengths of the interfacial interactions, but they are mere estimates. What does gel filtration say or cross linking? Does it fit with the biology/biochemistry expected of this protein? Anyway, have fun with your structure, but use a lot of skepticism in your interpretation. That will help you convince the reviewers. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.commailto:garav...@gmail.com On Dec 11, 2014, at 7:27 AM, Hay Dvir hd...@tx.technion.ac.ilmailto:hd...@tx.technion.ac.il 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 TechnionHaifa 323, Israel Tel: +(972)-77-887-1901 Fax: +(972)-77-887-1935 E-mail hd...@technion.ac.ilmailto:hd...@technion.ac.il Websitehttp://tcsb.technion.ac.ilhttp://tcsb.technion.ac.il/ To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing
Re: [ccp4bb] asymmetric homotrimer in the asu
Several years ago, we had an interesting case, where the molecule - a tetramer, did not possess the classical 222 or 4-fold symmetry. Rather, two monomers were related by a 2-fold, and other two monomers were related by yet another 2-fold. Ofcourse, the confirmation that it was indeed a tetramer was shown biochemically too! Our paper describing the open quaternary structure is: Banerjee et al., Proc Natl Acad Sci USA (1994) v. 91, 227- 231. Shekhar On Fri, Dec 12, 2014 at 12:30 AM, Oganesyan, Vaheh oganesy...@medimmune.com wrote: This fact by itself is unusual to say the least (for me): “ we have *NO* rotational symmetry (2, 3, or 4-fold) whatsoever between interacting monomers in the ASU or relating those built up by the crystallographic symmetry” There might be several ways of choosing molecules to represent the asymmetric unit. Is it possible to find ones that are related? Say something like non-crystallographic translation (or pseudo translation) + non-crystallographic rotation. For long time I was thinking about such a possibility of having more than one molecule in au but no rotation or pst. May be I’ve missed but never found an evidence, nor can I explain why would that be impossible. *Vaheh Oganesyan* *www.medimmune.com http://www.medimmune.com* *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Hay Dvir *Sent:* Thursday, December 11, 2014 12:04 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] asymmetric homotrimer in the asu Dear Michael, Thank you very much for the useful comments. Indeed, we are of course looking at it biochemically, which isn't a clear cut so far.. As you pointed out it could be a monomer in solution, but the interface between monomers within this asymmetric trimer seems too extensive (compared to those responsible for the lattice packing) not to suspect a trimer as a solution assembly. PISA suggested this asymmetric trimer as the most likely assembly but it falls into the grey region of their criteria (see attached pic.) Since it's rare, we are interested to know of other similar reports, if any, to learn how they were resolved/concluded. I believe the case you describe is not similar, as we have *NO* rotational symmetry (2, 3, or 4-fold) whatsoever between interacting monomers in the ASU or relating those built up by the crystallographic symmetry. Therefore I can't see how the space group information may help, but it is p212121 in case it helps boosting your morning coffee experience with symmetry pondering ... :). Cheers, Hay On Dec 11, 2014, at 3:47 PM, R. M. Garavito wrote: Dear Hay, And your point is? I am not trying to be snarky (although I am just starting my morning coffee), but to bring up the fact that CCP4BB readers need more info to comment on your case, like space group, local interactions, and how packed is tightly packed. I have had two cases of trimers, as my students initially called them, that were actually a dimer and a half. The half dimer had its mate in another ASU. Can it be a biological monomer that just happened to crystallize 3 monomers to an ASU? Non-symmetric homo-oligomers are rare, but sadly cannot be absolutely confirmed by crystallography alone, but by good old biochemistry. The PISA website ( http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) can give you estimations of the strengths of the interfacial interactions, but they are mere estimates. What does gel filtration say or cross linking? Does it fit with the biology/biochemistry expected of this protein? Anyway, have fun with your structure, but use a lot of skepticism in your interpretation. That will help you convince the reviewers. Cheers, Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry Molecular Biology* *603 Wilson Rd., Rm. 513 * *Michigan State University * *East Lansing, MI 48824-1319* *Office: (517) 355-9724 Lab: (517) 353-9125* *FAX: (517) 353-9334Email: rmgarav...@gmail.com garav...@gmail.com* ** On Dec 11, 2014, at 7:27 AM, Hay Dvir hd...@tx.technion.ac.il 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 TechnionHaifa 323, Israel Tel: +(972)-77-887-1901 Fax: +(972)-77-887-1935 E-mail hd...@technion.ac.il Website
Re: [ccp4bb] asymmetric homotrimer in the asu
Not sure if you are looking for something similar like this perhaps: http://www.rcsb.org/pdb/explore.do?structureId=2AUC Trimer in asu but all different. Jūrgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Streetx-apple-data-detectors://4, W8708 Baltimore, MD 21205x-apple-data-detectors://5/0 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.eduhttp://lupo.jhsph.edu/ On Dec 11, 2014, at 20:06, Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jpmailto:jt...@tsurumi.yokohama-cu.ac.jp 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 mechanicsand 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 DvirPh. D. HeadTechnion Center for Structural Biology TechnionHaifa 323, Israel Tel:+(972)-77-887-1901 Fax:+(972)-77-887-1935 E-mailhd...@technion.ac.ilmailto:hd...@technion.ac.il Websitehttp://tcsb.technion.ac.il
