<html><head></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><div>Hi Mark,</div><div><br></div><div>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. </div><div><br></div><div>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. </div><div><br></div><div>Finally, I would not trust SEC to differentiate between monomer and dimer - it is a shape sensitive tool.</div><div><br></div><div>I hope this is of some help.</div><div><br></div><div>Best wishes,</div><div>Olwyn</div><div><br></div><br><div><div>On 8 Jul 2011, at 11:08, Mark Agacan wrote:</div><br class="Apple-interchange-newline"><blockquote type="cite"><div style="MARGIN: 4px 4px 1px; FONT: 10pt Tahoma" text="#000000" bgcolor="#ffffff"><pre class="moz-signature" cols="72"><font face="Tahoma">Hello RASMB,</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">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.</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">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.</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">However, the crystal structure, SEC, etc. indicate this protein should be a <em>dimer</em>.</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">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</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">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</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">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.</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">Would you, on the basis of RMSD values, assign monomer or dimer to this peak?</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">Thanks,</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma">Mark</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma"></font> </pre><pre class="moz-signature" cols="72"><font face="Tahoma">~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~<br>Dr Mark Agacan, Scientific Officer for the Division of Biological Chemistry and Drug Discovery,<br>Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH <br>Tel: +44 1382 386095 Fax: +44 1382 345764 Mobile: 07525 451 117<br>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~</font></pre><pre class="moz-signature" cols="72"><font face="Tahoma"></font> </pre>
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<span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Courier; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; font-size: medium; "><span class="Apple-style-span" style="border-collapse: separate; color: rgb(0, 0, 0); font-family: Courier; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; -webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; font-size: medium; "><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">________________________________________________________________<br>Olwyn Byron<br><br>School of Life Sciences<br>College of Medical, Veterinary & Life Sciences<br>Sir Graeme Davies Building, University of Glasgow<br>120 University Place, Glasgow G12 8QQ, Scotland, UK<br><br>+44 (0)141 330 3752 or +44 (0)7575 766462<br><a href="mailto:olwyn.byron@glasgow.ac.uk">olwyn.byron@glasgow.ac.uk</a><br>http://www.gla.ac.uk:443/ibls/staff/staff.php?who=Pn||ed<br>________________________________________________________________<br>The University of Glasgow, charity number SC004401</div></span></span>
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