[ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
the tandem KH domain of FMRP crystallized as a very convincing dimer (valverde et al 2007), but is a monomer in solution, although it is not the whole protein but just two domains of it.. anyway, i would think these ar much more common than the other way around. Tommi Quoting Poul Nissen [EMAIL PROTECTED]: I would say that all crystals represent hyper-oligomeric structures, but never mind, I know what you mean ;-) the E. coli EF-Tu:EF-Ts complex is a good example - the structure clearly indicates an (EF-Tu:EF-Ts)2 dimer, and the T. thermophilus EF-Tu:EF-Ts is even a disulphide-linked dimer. However, all solution studies indicate that the E.coli EF-Tu:EF-Ts complex is in fact a monomeric complex. Poul On 11/12/2008, at 17.09, Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
On Thursday 11 December 2008, Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? I don't think such a question is entirely well-defined, for two reasons. 1) The monomer/dimer equilibrium in solution may well depend on the specific conditions (pH, concentration, presence of ligands, temperature, etc). Unless these conditions are replicated in your crystallization medium, it is uncertain to what extent the solution measurement is relevant. 2) How extensive an interface is required in order for it to be considered a dimer/multimer interaction? In the limiting case of very small interfaces, the entire crystal might be consider a single oligomer, with each lattice-packing contact constituting a monomer:monomer interaction. That's not a very useful place to set the threshold, but where do you set it - 100 A^2 ? 500 A^2 ? 1000 A^2? Some definition other than surface area? That said, I have some interest in the question as a practical matter. We have a new structure that is obviously, but totally unexpectedly, a tetramer in the crystal. In this case the monomer:monomer interaction surface is 1500 A^2. But exactly what criteria would I use to argue that this is a real tetramer? What criteria would I use to argue that it is a crystal artifact? Yes, of course ideally one would go back to the lab and survey for solution measurements that are consistent with tetramerization, but that is not always practical, and may lead right back to your original question. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] [SPAM:#] [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Mass action is on the crystal's side. Two recent examples of proteins that are dimers by standard solution assays, but form weak/transient/co-factor-dependent tetramers to function, and those tetramers are seen in the crystal. (There is good solution data to back up the relevance of the tetramer in both cases). Yuan P, Gupta K, Van Duyne GD. Tetrameric structure of a serine integrase catalytic domain. Structure. 2008 Aug 6;16(8):1275-86. Mouw KW, Rowland SJ, Gajjar MM, Boocock MR, Stark WM, Rice PA. Architecture of a serine recombinase-DNA regulatory complex. Mol Cell. 2008 Apr 25;30(2):145-55. Phoebe == Original message Date: Thu, 11 Dec 2008 10:09:33 -0600 From: Santarsiero, Bernard D. [EMAIL PROTECTED] Subject: [SPAM:#] [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? To: CCP4BB@JISCMAIL.AC.UK In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] [SPAM:#] [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Here we are dealing with two different state of chemistry, solid state and solution state, If one of the minima in solid state resembles the biological state minimum, then there is a possiblw way to clearly define the biology and its significant interaction of that particular 'mer' of a protein, other wise we end up with pure physical interaction. But my question is have we answered Wouldn't the high concentration in the crystallization drop further favor dimerization? this part ... S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany. On Thu, Dec 11, 2008 at 5:53 PM, Phoebe Rice [EMAIL PROTECTED] wrote: Mass action is on the crystal's side. Two recent examples of proteins that are dimers by standard solution assays, but form weak/transient/co-factor-dependent tetramers to function, and those tetramers are seen in the crystal. (There is good solution data to back up the relevance of the tetramer in both cases). Yuan P, Gupta K, Van Duyne GD. Tetrameric structure of a serine integrase catalytic domain. Structure. 2008 Aug 6;16(8):1275-86. Mouw KW, Rowland SJ, Gajjar MM, Boocock MR, Stark WM, Rice PA. Architecture of a serine recombinase-DNA regulatory complex. Mol Cell. 2008 Apr 25;30(2):145-55. Phoebe == Original message Date: Thu, 11 Dec 2008 10:09:33 -0600 From: Santarsiero, Bernard D. [EMAIL PROTECTED] Subject: [SPAM:#] [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? To: CCP4BB@JISCMAIL.AC.UK In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
There are a number of examples of nuclear receptor heterodimers, where crystallization of the individual partner, such as PPAR or LXR, crystallizes as a homodimer, even though these species do not exist in solution. There are also many examples of dimers showing one molecule per asymmetric unit, but the physiological dimer is apparent in the crystal packing. Kendall Nettles On 12/11/08 11:09 AM, Santarsiero, Bernard D. [EMAIL PROTECTED] wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Hi Bernie, We had a case recently which was a dimer in the crystal (with 2 Ca binding sites in the symmetric dimer interface) but anSEC gave monomer under standard conditions ( 20mM Tris, 200mM NaCl, 0.5mM TCEP at pH7.5, Temperature at 8C ). The crystals had 0.2 M Ca Acetate. We had a little protein left over and tried running anSEC+SLS after adding Ca2+ to the protein sample and using a mobile phase of 20 mM Tris pH 7.5, 200 mM NaCl, 0.2 M CaCl2. It then ran as a dimer. See comments in remark 300 for pdb id 3DB7 http://www.pdb.org/pdb/explore/explore.do?structureId=3DB7 and the related TOPSAN page for this protein -- http://www.topsan.org/explore?pdbId=3db7 This supports Pat Loll Ethan Merritt's comments about the conditions (crystal and anSEC) influencing on the oligomerizaiton state. Neither of these conditions are what we expect the protein sees in the periplasm and we did not any protein left to investigate the concentration of Ca needed to shift the distribution from monomer to dimer, so it is hard to say for sure how it functions physiologically. Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Ethan A Merritt Sent: Thursday, December 11, 2008 8:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? On Thursday 11 December 2008, Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? I don't think such a question is entirely well-defined, for two reasons. 1) The monomer/dimer equilibrium in solution may well depend on the specific conditions (pH, concentration, presence of ligands, temperature, etc). Unless these conditions are replicated in your crystallization medium, it is uncertain to what extent the solution measurement is relevant. 2) How extensive an interface is required in order for it to be considered a dimer/multimer interaction? In the limiting case of very small interfaces, the entire crystal might be consider a single oligomer, with each lattice-packing contact constituting a monomer:monomer interaction. That's not a very useful place to set the threshold, but where do you set it - 100 A^2 ? 500 A^2 ? 1000 A^2? Some definition other than surface area? That said, I have some interest in the question as a practical matter. We have a new structure that is obviously, but totally unexpectedly, a tetramer in the crystal. In this case the monomer:monomer interaction surface is 1500 A^2. But exactly what criteria would I use to argue that this is a real tetramer? What criteria would I use to argue that it is a crystal artifact? Yes, of course ideally one would go back to the lab and survey for solution measurements that are consistent with tetramerization, but that is not always practical, and may lead right back to your original question. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
On Thu, Dec 11, 2008 at 8:09 AM, Santarsiero, Bernard D. b...@uic.eduwrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? There are several families of receptor kinases that behave like this, specifically EGFR in humans and some of the Ser/Thr kinases in M. tuberculosis. The kinase domains alone have very low (millimolar) affinity but dimerization in response to an extracellular signal is required for activation. In both cases the activation mechanisms were not understood until the dimeric crystal structures were (accidentally) obtained, and were later confirmed by biochemical experiments: http://www.ncbi.nlm.nih.gov/pubmed/16777603 (PDB IDs: 2gs2, 2gs6, 2gs7) http://www.ncbi.nlm.nih.gov/pubmed/17242402 (and cited papers - PDB IDs: 1mru, 1o6y, 2fum, 2h34)
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero Sometimes proteins are (mostly) monomers in solution and become domain-swapped trimers in the crystal. Zegers et all, PNAS 1999 Feb 2;96(3):818-22 kind regards, Klaas
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
...and in absence of TM domains and the 2D restriction of the membrane they will probably not dimerize as free domains in solution now suddenly gained the freedom of 3D diffusion On 11/12/2008, at 19.03, Nathaniel Echols wrote: On Thu, Dec 11, 2008 at 8:09 AM, Santarsiero, Bernard D. b...@uic.edu wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? There are several families of receptor kinases that behave like this, specifically EGFR in humans and some of the Ser/Thr kinases in M. tuberculosis. The kinase domains alone have very low (millimolar) affinity but dimerization in response to an extracellular signal is required for activation. In both cases the activation mechanisms were not understood until the dimeric crystal structures were (accidentally) obtained, and were later confirmed by biochemical experiments: http://www.ncbi.nlm.nih.gov/pubmed/16777603 (PDB IDs: 2gs2, 2gs6, 2gs7) http://www.ncbi.nlm.nih.gov/pubmed/17242402 (and cited papers - PDB IDs: 1mru, 1o6y, 2fum, 2h34)
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
I wanted to comment on a couple of things that came up during this discussion. 1. We use crystallography because it enables us to get structural information. But we have to be aware that most of the time a crystal will not be an exact reflection of the biological environment, which is usually what we want to relate our structure to. For this reason, to make any useful interpretations of the oligomeric state and how it relates to a biologically relevant situation, one needs to complement it with other studies (hopefully in a solution that better resembles the biological situation). 2. What you find in the asymmetric unit of a crystal does not necessarily have anything to do with the biologically relevant oligomeric state (biological unit). I commonly see researchers confuse the asymmetric unit with the biological unit, even in submitted and published papers. To phrase it in a different way, crystallographic symmetry often relate subunits in an oligomer, and conversely NCS often corresponds to just another biologically irrelevant crystal contact. 3. It is often not trivial to distinguish crystal contact from the oligomeric interface. There is lots of literature on this, and software and databases that can help you distinguish between these (using the size of interface area, but also many other criteria to sort these interactions). It is often not going to be possible to do so with any high reliability without complementary experiments. Please refer to the references cited in this recent conference proceeding for extensive literature on this matter: http://www.ncbi.nlm.nih.gov/pubmed/19021571?ordinalpos=1itool=EntrezSystem2 .PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSu m. Best wishes Bostjan On 12/12/08 2:34 AM, Ethan A Merritt merr...@u.washington.edu wrote: On Thursday 11 December 2008, Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? I don't think such a question is entirely well-defined, for two reasons. 1) The monomer/dimer equilibrium in solution may well depend on the specific conditions (pH, concentration, presence of ligands, temperature, etc). Unless these conditions are replicated in your crystallization medium, it is uncertain to what extent the solution measurement is relevant. 2) How extensive an interface is required in order for it to be considered a dimer/multimer interaction? In the limiting case of very small interfaces, the entire crystal might be consider a single oligomer, with each lattice-packing contact constituting a monomer:monomer interaction. That's not a very useful place to set the threshold, but where do you set it - 100 A^2 ? 500 A^2 ? 1000 A^2? Some definition other than surface area? That said, I have some interest in the question as a practical matter. We have a new structure that is obviously, but totally unexpectedly, a tetramer in the crystal. In this case the monomer:monomer interaction surface is 1500 A^2. But exactly what criteria would I use to argue that this is a real tetramer? What criteria would I use to argue that it is a crystal artifact? Yes, of course ideally one would go back to the lab and survey for solution measurements that are consistent with tetramerization, but that is not always practical, and may lead right back to your original question. --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html Office: Building 76 Room 452 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland.
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
I recently had a case, unpublished right now, where the NMR structure of the monomer was determined and all other biochemical evidence showed a monomer as the active form. The resulting crystal structure turned out to be a domain swapped dimer. The group I did the work for are still puzzling over it and have tried a number of things to see if the structure is biologically significant. Leonard Thomas Ph. D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73032 lmtho...@ou.edu Office: 405-325-1126 Lab: 405-325-7571 On Dec 11, 2008, at 10:09 AM, Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? [SEC=UNCLASSIFIED]
On the question of solution structure vs crystal structure, another comment worth considering is this: The crystalline state of a protein can in some cases (eg a large enzyme with small substrates) be more similar to physiological conditions than a dilute solution. If crowding effects are important, the crystalline state, at ~50% (w/v), is well crowded, unlike typical solution studies that are done at concentrations more like 1% (w/v) and below. David Goodsell's illustrations (http://mgl.scripps.edu/people/goodsell/) may be illuminating here. I also find that non-crystallographers need to be told that protein crystal contacts are weak and tenuous, and I find it useful to show a cross-section of a crystal, whether showing modelled protein or electron density of multiple repeats of the unit cell. Anthony Anthony DuffTelephone: 02 9717 3493 Mob: 043 189 1076 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Bostjan Kobe Sent: Friday, 12 December 2008 10:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? I wanted to comment on a couple of things that came up during this discussion. 1. We use crystallography because it enables us to get structural information. But we have to be aware that most of the time a crystal will not be an exact reflection of the biological environment, which is usually what we want to relate our structure to. For this reason, to make any useful interpretations of the oligomeric state and how it relates to a biologically relevant situation, one needs to complement it with other studies (hopefully in a solution that better resembles the biological situation). 2. What you find in the asymmetric unit of a crystal does not necessarily have anything to do with the biologically relevant oligomeric state (biological unit). I commonly see researchers confuse the asymmetric unit with the biological unit, even in submitted and published papers. To phrase it in a different way, crystallographic symmetry often relate subunits in an oligomer, and conversely NCS often corresponds to just another biologically irrelevant crystal contact. 3. It is often not trivial to distinguish crystal contact from the oligomeric interface. There is lots of literature on this, and software and databases that can help you distinguish between these (using the size of interface area, but also many other criteria to sort these interactions). It is often not going to be possible to do so with any high reliability without complementary experiments. Please refer to the references cited in this recent conference proceeding for extensive literature on this matter: http://www.ncbi.nlm.nih.gov/pubmed/19021571?ordinalpos=1itool=EntrezSys tem2 .PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVD ocSu m. Best wishes Bostjan On 12/12/08 2:34 AM, Ethan A Merritt merr...@u.washington.edu wrote: On Thursday 11 December 2008, Santarsiero, Bernard D. wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? I don't think such a question is entirely well-defined, for two reasons. 1) The monomer/dimer equilibrium in solution may well depend on the specific conditions (pH, concentration, presence of ligands, temperature, etc). Unless these conditions are replicated in your crystallization medium, it is uncertain to what extent the solution measurement is relevant. 2) How extensive an interface is required in order for it to be considered a dimer/multimer interaction? In the limiting case of very small interfaces, the entire crystal might be consider a single oligomer, with each lattice-packing contact constituting a monomer:monomer interaction. That's not a very useful place to set the threshold, but where do you set it - 100 A^2 ? 500 A^2 ? 1000 A^2? Some definition other than surface area? That said, I have some interest in the question as a practical matter. We have a new structure that is obviously, but totally unexpectedly, a tetramer in the crystal. In this case the monomer:monomer interaction surface is 1500 A^2. But exactly what criteria would I use to argue that this is a real tetramer? What criteria would I use to argue that it is a crystal artifact? Yes, of course ideally one would go back to the lab and survey for solution measurements that are consistent with tetramerization, but that is not always practical, and may lead right back to your original question. --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience Cooper Road University of Queensland Brisbane,
Re: [ccp4bb] [SPAM:#] [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
I would strongly argue against protein crystals (in most cases) being solid state. Most of the surface of a molecule is actually solvated and protein crystals as they are miss some of the typical properties of solid state. Although in some cases oligomerization occuring upon protein crystallization indicates fairly strong interactions between molecules, still the crystals are actually half liquid. This is the main reason why many ligand exchange and activity studies could be performed even in protein crystals. Quite often (I think) protein crystallographers would actually like their crystals to really behave like solids (stability, localization of disordered regions, etc.) no need for cryoprotection and it is hard to make them ... Jan Dohnalek IMC Prague Jayashankar wrote: Here we are dealing with two different state of chemistry, solid state and solution state, If one of the minima in solid state resembles the biological state minimum, then there is a possiblw way to clearly define the biology and its significant interaction of that particular 'mer' of a protein, other wise we end up with pure physical interaction. But my question is have we answered Wouldn't the high concentration in the crystallization drop further favor dimerization? this part ... S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany. On Thu, Dec 11, 2008 at 5:53 PM, Phoebe Rice pr...@uchicago.edu mailto:pr...@uchicago.edu wrote: Mass action is on the crystal's side. Two recent examples of proteins that are dimers by standard solution assays, but form weak/transient/co-factor-dependent tetramers to function, and those tetramers are seen in the crystal. (There is good solution data to back up the relevance of the tetramer in both cases). Yuan P, Gupta K, Van Duyne GD. Tetrameric structure of a serine integrase catalytic domain. Structure. 2008 Aug 6;16(8):1275-86. Mouw KW, Rowland SJ, Gajjar MM, Boocock MR, Stark WM, Rice PA. Architecture of a serine recombinase-DNA regulatory complex. Mol Cell. 2008 Apr 25;30(2):145-55. Phoebe == Original message Date: Thu, 11 Dec 2008 10:09:33 -0600 From: Santarsiero, Bernard D. b...@uic.edu mailto:b...@uic.edu Subject: [SPAM:#] [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp -- == Mr. Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Laboratory of Structural Analysis of Molecules Heyrovskeho nam. 2 16206 Prague 6 Tel: +420 296809390 Fax: +420 296809410 http://protein.awardspace.com/ ==
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Hi Thierry, We have worked on a number of proteins where the oligomerisation state differs from that found in solution. A recent example is a project we worked on involving the HC fragment of tetanus toxin (see Qazi et al., J Mol Biol. 2007 Jan 5;365(1):123-34) which crystallises as a monomer. The discovery of it multimerising was somewhat serendipitous in that the protein (like so many these days) was purified using a his-tag and then crystallised. We planned to use SAXS to study proposed conformational changes and looked at the protein on a native gel as part of our standard preparation for these experiments. The native gel showed the protein multimerised and the combination of the SAXS and excellent crystal structure from the Isaacs group led to new ideas about the function of the toxin. A few points: 1. Crystallisation conditions can be very selective for different oligomerisation states of protein and other parameters ( e.g., pH, ionic strength, oxidation state, exogenous ligands,...) besides concentration can affect the equilibria controlling whether a protein appears to be a monomer or not as the case may be. 2. It is always worth looking carefully at native gels or other sizing data for any protein which is crystallised. With the great expression systems out there using affinity tags this sometimes gets forgotten. 3. Be aware that truncated forms of proteins (even small deletions) may affect oligomerisation states (we have also seen this with a membrane bound receptor we are working with now and I think there are other similar examples in the literature) best wishes, Kate
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
hi thierry we had a case where Spo0A in the phospho form was a dimer in solution, and in the non-phospho form was a monomer. in the crystal, we had monomers of the phospho form, and (domain-swapped) dimers of the non-phospho form. turns out that the crystallisation conditions affected the behaviour of the protein, converting a M-D Kd from nanomolar to micromolar in the first instance, and the acid pH of the crystallisation solution in the latter promoted domain- swapping. the biochemistry where we sorted all this out is: http://www.sciencedirect.com/science?_ob=ArticleURL_udi=B6WK7-457CYHW-HN_user=224739_rdoc=1_fmt=_orig=search_sort=dview=c_acct=C14659_version=1_urlVersion=0_userid=224739md5=5d3b66d5d8d662753ec43bc94a173880 rick Fischmann, Thierry wrote: Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn’t the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete. -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 222 5482 University of Newcastle Fax: +44 (0)191 222 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: [EMAIL PROTECTED]
[ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn't the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete.
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Thank you Patrick for the reply, as well to another person who has replied directly to me. Please provide with examples if you know of any (say a reference or a PDB id ), as it would allow for comparison between published results and my own crystallization system. To answer your points: The crystallization conditions in the case at hand are as follow : the precipitant is PEG, the amount of salt is relatively low (0.1 M buffer + some NaCl etc.), and the pH is 8.0. The SEC experimental conditions are not too far away from the crystallization conditions except, of course, for the presence of PEG. Thierry From: Patrick Loll [mailto:[EMAIL PROTECTED] Sent: Monday, December 01, 2008 05:59 PM To: Fischmann, Thierry Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer? depends on the crystallization conditions (and the SEC conditions). E.g., if the crystals grow in 4 M salt at pH 3, maybe that disrupts a complex that forms under more physiological conditions On 1 Dec 2008, at 5:47 PM, Fischmann, Thierry wrote: Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn't the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete. --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete.
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
Yo Thierry: The periplasmic domain of the aspartate receptor, in the absence of ligand, 1lih, is a dimer, but crystallizes as a monomer in the sense that there is one monomer per asymmetric unit. There is a disulphide bond between two Cys36 that maintains it as a dimer (and indeed reduction of this bond inhibits crystallization). Each of two ligand binding sites spans both monomers. So based on that, the biologically relevant form is definitely a dimer, so you can't conclude otherwise based on the fact that it crystallizes as one monomer per asymmetric unit. Now if it were to crystallize in a space group lacking a crystallographic 2-fold coincident with the natural dimer axis, that might be a different story. When you add aspartate, it crystallizes as a dimer with only one of two potential binding sites occupied by the ligand. Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Dec 1, 2008, at 2:47 PM, Fischmann, Thierry wrote: Dear fellow crystallographers, This is a question which is not CCP4-related. Is anybody aware of a protein which is known to be a dimer in solution (say by SEC), and yet crystallizes as a monomer? Wouldn't the high concentration in the crystallization drop further favor dimerization? In other words, if a protein crystallizes as a monomer, can I conclude that it does not form biologically relevant dimers in solution? Thank you in advance for your replies. Thierry * This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete.
