mea culpa! How about FRET? JPK
On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen <jubo...@jhsph.edu> 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 <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 > ******************************************* > > ...................... > 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: j-kell...@northwestern.edu *******************************************
