[RASMB] sedfit vs SOMO vs structure

[Borries Demeler]

> 
> Hello RASMB,Sedfit c(s) analysis of a protein I'm working with shows a 30 =
> kDa peak with S =3D 2.60 and f/f0 of 1.2, that corresponds to monomer.SOMO =
> in Ultrascan using the pdb file for this protein predicts a 30 kDa monomer =
> at 2.42, with f/f0 of 1.19.  It's good agreement with the sedimentation =
> velocity results.However, the crystal structure, SEC, etc. indicate this =
> protein should be a dimer.Starting with the default value of 1.2 for the =
> frictional ratio and floating in sedfit returns a value of 1.2, with MW =
> that corresponds to monomer.  RMSD =3D 0.009756Doubling this value to 2.4 =
> and floating gives a value of 2.3, and assigns a mass that corresponds to =
> dimer to the main peak in the c(s) analysis.  RMSD =3D 0.009324Thus there =
> appear to be two energy minima for the frictional ratio, depending which =
> initial value is used.  The RMSD value is lower when the higher value of =
> f/f0 is used.Would you, on the basis of RMSD values, assign monomer or =
> dimer to this peak?Thanks,Mark ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~=
> ~~~~~~~~~~~~~
> Dr Mark Agacan, Scientific Officer for the Division of Biological =
> Chemistry and Drug Discovery,
> Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, =
> Dundee, DD1 5EH=20
> Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~=20

Mark, is this absorbance or interference data? If absorbance data I
find the RMSD values about 2-3 times too high to be representative. So I
would question the quality of the data or the fit, or both. On absorbance
data we typically get about 2e-3 - 3e-3 for RMSD at 280 and 230 nm. Do
you get random residuals throughout the fit? If so, I would check the
instrument. If the fits doesn't produce random residuals, I would question
your fit and model.

Regards, -Borries