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 <p...@mrc-lmb.cam.ac.uk> 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: j-kell...@northwestern.edu *******************************************
