Free flow electrophoresis would be another option, by the way anybody on the East Coast who has one of those instruments ? I'd be interested to get an email directly.
Thanks, Jürgen On Sep 5, 2011, at 1:00 PM, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen <[email protected]<mailto:[email protected]>> wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans <[email protected]<mailto:[email protected]>> wrote: I get confused by these figures. As I understand it the "interface area" given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the "real" dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [email protected]<mailto:[email protected]> ******************************************* ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [email protected]<mailto:[email protected]> ******************************************* ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
