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.


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 
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,

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