[RASMB] sedfit vs SOMO vs structure

[Olwyn Byron]

Hi Mark,

An f/f0 of 2.3 corresponds to an axial ratio of about 30:1 for a roughly prolate (anhydrous) molecule (50:1 if it is more oblate).  I'm guessing, from your SOMO hydrodynamics computations for the monomer, that (even allowing for hydrodynamic hydration) there is no way your dimer could have this axial ratio.

Also, have you used SOMO to compute the sedimentation coefficient for the crystallographic dimer?  How does this compare with your experimentally determined s ?  Obviously a putative solution dimer need not be equivalent to the crystallographic form, but it's a starting point.

Finally, I would not trust SEC to differentiate between monomer and dimer - it is a shape sensitive tool.

I hope this is of some help.

Best wishes,
Olwyn


On 8 Jul 2011, at 11:08, 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?

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
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~



************************************************************
Please consider the environment. Do you really need to print this email?


The University of Dundee is a registered Scottish charity, No: SC015096

_______________________________________________
RASMB mailing list
RASMB at rasmb.bbri.org<mailto:RASMB at rasmb.bbri.org>
http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb

________________________________________________________________
Olwyn Byron

School of Life Sciences
College of Medical, Veterinary & Life Sciences
Sir Graeme Davies Building, University of Glasgow
120 University Place, Glasgow G12 8QQ, Scotland, UK

+44 (0)141 330 3752 or +44 (0)7575 766462
olwyn.byron at glasgow.ac.uk<mailto:olwyn.byron at glasgow.ac.uk>
http://www.gla.ac.uk:443/ibls/staff/staff.php?who=Pn||ed
________________________________________________________________
The University of Glasgow, charity number SC004401

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20110708/59c6e0af/attachment-0001.html>