Re: [ccp4bb] Binding constants/kinetics for crystallisation
Good points have been brought up; here's one more to consider from my experience. If you are going to run SEC prior to crystallization, I would highly recommend running a native gel of the peak you collect. Especially if you don't know the stoichiometry or if the stoichiometry is complex. I crystallized a multimeric complex where a large oligomer bound to four smaller proteins with high affinity (sub nM), and even after making the complex in stoichiometric excess, somehow one of the ligands would fall off during SEC (probably the 3-bound and 4-bound mers were in equilibrium on column) and only after I discovered this by running a native gel did I start getting single crystals instead of multi-lattice xtals. Basically the band for the full complex was fuzzy (I.e. A range of stoichiometries) and after titrating in more ligand after SEC did the band look nice. Also, you can run a native gel of your crystals (assuming you obtain some!) by harvesting a number of them in a buffer in which they'll dissolve and then use a sensitive staining method, like silver staining. Good luck! Geoff On 12/7/12 4:00 PM, CCP4BB automatic digest system lists...@jiscmail.ac.uk wrote:
[ccp4bb] Binding constants/kinetics for crystallisation
Dear all - is there a rule of thumb for favourable values of Kd, kon and koff of protein-protein or protein-dna complexes for protein crystallisation? Are these measurements useful in crystallisation, or should one just put it down a gel filtration column, hope for a complex and not worry? If anyone can point toward a reference, i'd appreciate it. Roger
Re: [ccp4bb] Binding constants/kinetics for crystallisation
Dear Roger, In my humble opinion, the qualitative knowledge that the complex actually forms (established through pull down assays, gel filtration etc) is probably far more important than the Kd values in solution. In any case, the crystallization is done at very high concentrations, far above the Kd values of the interacting partners. So having a thumb rule is not necessary. The knowledge of the stoichiometry of the binding partners may actually be more important to get a more homogenious complex. In a recent complex I've worked on, we determined the Kd only after solving the structure. . cheers Ganesh Le 07/12/12 16:11, Roger Pickman a écrit : Dear all - is there a rule of thumb for favourable values of Kd, kon and koff of protein-protein or protein-dna complexes for protein crystallisation? Are these measurements useful in crystallisation, or should one just put it down a gel filtration column, hope for a complex and not worry? If anyone can point toward a reference, i'd appreciate it. Roger
Re: [ccp4bb] Binding constants/kinetics for crystallisation
Dear Roger, I disagree with Ganesh. Knowing the stoichiometry is not necessary. Stoichiometry may need adjusting to reflect the relative solubility of the interacting partners under the various crystallization conditions. See also: Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G., Silverman, G.J. Charbonnier, J.-B. (2001) Crystallization of macromolecular complexes: Stoichiometric variation screening. J. Cryst. Growth 232: 580-590. Affinity is very important when the affinity is better than nanomolar, you do not need stoichiometric compensation. Enrico. On Fri, 07 Dec 2012 16:44:48 +0100, Ganesh Natrajan ganesh.natra...@ibs.fr wrote: Dear Roger, In my humble opinion, the qualitative knowledge that the complex actually forms (established through pull down assays, gel filtration etc) is probably far more important than the Kd values in solution. In any case, the crystallization is done at very high concentrations, far above the Kd values of the interacting partners. So having a thumb rule is not necessary. The knowledge of the stoichiometry of the binding partners may actually be more important to get a more homogenious complex. In a recent complex I've worked on, we determined the Kd only after solving the structure. . cheers Ganesh Le 07/12/12 16:11, Roger Pickman a écrit : Dear all - is there a rule of thumb for favourable values of Kd, kon and koff of protein-protein or protein-dna complexes for protein crystallisation? Are these measurements useful in crystallisation, or should one just put it down a gel filtration column, hope for a complex and not worry? If anyone can point toward a reference, i'd appreciate it. Roger -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Binding constants/kinetics for crystallisation
Roger, Very low values of Kd (nM) may mean that you have a good chance of finding both the proteins (or protein-nucleic acid) together if you get crystals of the mixture. I therefore think that the measurements are useful in that sense. However, low Kd does not necessarily mean that you get the complex to crystallize. It is possible that one of the components packs better, therefore would crystallize on its own. Also, there could be cases where the proteins have very low affinity for each other (Kd in 100's of micromolar to millimolar range), yet would readily crystallize as a complex. If you want to know some specific cases in this category, look for examples of some ubiquitin receptors co-crystallized with ubiquitin. Gel filtration of the mixture is not the only answer. You could also mix A and B with one of the components in slight excess (say B is 1.5-fold molar excess than A) and then set up trays without any chromatography. I have spoken to a few crystallographers working on protein-protein complex and they all seem to agree that the the mixing approach works. I am not sure if anyone has studied the relationship between thermodynamics of protein-protein or protein-nucleic acid complex and their crystallizability. I would like to know, if you find any. Chitta - Original Message - From: Roger Pickman rpick...@googlemail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, December 7, 2012 10:11:15 AM Subject: [ccp4bb] Binding constants/kinetics for crystallisation Dear all - is there a rule of thumb for favourable values of Kd, kon and koff of protein-protein or protein-dna complexes for protein crystallisation? Are these measurements useful in crystallisation, or should one just put it down a gel filtration column, hope for a complex and not worry? If anyone can point toward a reference, i'd appreciate it. Roger
