[RASMB] sedfit vs SOMO vs structure

[Ewa Folta-Stogniew]

At 06:08 AM 7/8/2011, Mark Agacan wrote:

>Hello RASMB,
>Sedfit c(s) analysis of a protein I'm working with shows a 30 kDa 
>peak with S = 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 = 0.009756
>Doubling 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 = 0.009324
>Thus 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?

I would run SEC/MALLS with DLS on line- to get direct measurement of 
MW and Rh. which will allow you to determine f/fo value. .

See attached.

Ewa


>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
>Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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