Hi Sebastiano, I don't see how the k-off would influence this, given the timescale of growing crystals. An explanation in terms of high Kd and relative lack of crystal contacts for the component with higher temperature factors would sound more convincing to me. Mark
Quoting Vellieux Frederic: > I can direct you to PDB entry 1EWY, where the average isotropic > temperature factor for the major component of the complex is ca. 47 > A**2 and that for the smaller component is ca. 69 A**2. Similar > values than the ones you are reporting. I am assuming some sort of > "disorder", or if you prefer, "wobbling" of the smaller component at > the lever of the binding site. > > Fred. > > Sebastiano Pasqualato wrote: >> Hi all, >> I have a crystallographical/biochemical problem, and maybe some of >> you guys can help me out. >> >> We have recently crystallized a protein:protein complex, whose Kd >> has been measured being ca. 10 uM (both by fluorescence polarization >> and surface plasmon resonance). >> Despite the 'decent' affinity, we couldn't purify an homogeneous >> complex in size exclusion chromatography, even mixing the protein at >> concentrations up to 80-100 uM each. >> We explained this behavior by assuming that extremely high Kon/Koff >> values combine to give this 10 uM affinity, and the high Koff value >> would account for the dissociation going on during size exclusion >> chromatography. We have partial evidence for this from the SPR >> curves, although we haven't actually measured the Kon/Koff values. >> >> We eventually managed to solve the crystal structure of the complex >> by mixing the two proteins (we had to add an excess of one of them >> to get good diffraction data). >> Once solved the structure (which makes perfect biological sense and >> has been validated), we get mean B factors for one of the component >> (the larger) much lower than those of the other component (the >> smaller one, which we had in excess). We're talking about 48 Å^2 vs. >> 75 Å^2. >> >> I was wondering if anybody has had some similar cases, or has any >> hint on the possible relationship it might (or might not) exist >> between high a Koff value and high B factors (a relationship we are >> tempted to draw). >> >> Thanks in advance, >> best regards, >> ciao >> s >> >> >> > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
