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<DIV>Chad</DIV>
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<DIV>This requires a discussion of error analysis and the propagation of error. If you really are getting a 21/24 reduction in MW that is 12.5% too low. This might be due to an error in vbar*rho - I presume you are also calculating the buffer density, but lets assume its all in vbar - at a vbar of 0.75 that would correspond to an error in vbar of ~4.2%. This seems like a larger error in a calculated vbar but it really depends upon the solvent and what might be binding to the protein to alter it. Alternatively, its a volume change upon assembly & thus pressure dependent. ATCase is the classic example of this and in that case its S value changes by 3% (upon ligand binding) & thus a 1% change in vbar. Another example is myosin assembly, but that system exhibits a remarkable P-dependent disassembly during sedimentation.</DIV>
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<DIV>Of course the whole question depends upon the assumption that its a tight complex and not exhibiting boundary spreading due to the fact its an interacting system. You get a low MW whenever you have a reaction boundary due to the spreading of the "peak" - think D increasing in an s/D calculation and thus MW decreases. Does the data exhibit concentration dependence? Spreading can also occur if the system exhibits kinetic effects. </DIV>
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<DIV>-------------------------------------------------------------------<BR> Dr. John J. "Jack" Correia<BR> Department of Biochemistry<BR> University of Mississippi Medical Center<BR> 2500 North State Street<BR> Jackson, MS 39216<BR> (601) 984-1522 <BR> fax (601) 984-1501 <BR> email address: <A href="mailto:jcorreia@biochem.umsmed.edu">jcorreia@biochem.umsmed.edu</A> <BR> homepage location: <A href="http://biochemistry.umc.edu/correia.html">http://biochemistry.umc.edu/correia.html</A><BR> dept homepage location: <A href="http://biochemistry.umc.edu/">http://biochemistry.umc.edu/</A><BR>-------------------------------------------------------------------<BR> <BR> <BR><BR><BR>>>> Chad Brautigam <Chad.Brautigam@UTSouthwestern.edu> 06/06/06 1:45 PM >>><BR></DIV>
<DIV style="COLOR: #000000">Hello, All,<BR><BR>Sorry if this is a rudimentary question. I have been using velocity <BR>sedimentation to examine the oligomeric states of a protein and <BR>mutants thereof. Some mutants are trimers, and the molecular weight <BR>estimates given by sedfit (either a c(M) distribution or a discrete <BR>species model) are very reasonable. However, based on a crystal <BR>structure, we expect the wild-type to be a 24-mer. Sedfit <BR>consistently underestimates the molecular weight ( I get something <BR>more akin to 21-mer).<BR><BR>I assume that there are at least to possibilites here:<BR><BR>1. The crystal structure is wrong, and the thing really is a 21-mer <BR>in solution.<BR><BR>2. The vbar calculated by Sednterp is inaccurate- it is not <BR>accounting for the fact that some of the volume of the 24-mer is not <BR>taken up by protein, but by solvent. The vbar is therefore <BR>significantly too low, with adverse effects on the MW calculation.<BR><BR>Does anyone know if there is a more accurate way to estimate vbar in <BR>cases of large macromolecular assemblies? Can our crystal structure <BR>help us out in any way?<BR><BR>BTW, yes, I know that sed. equilibrium might be the preferred <BR>approach in this case, but instrument time is limited at the moment.<BR><BR>Thanks,<BR>Chad<BR><BR><BR>==================================<BR>Chad A. Brautigam, Ph.D.<BR>Research Scientist<BR>The University of Texas<BR>Southwestern Medical Center at Dallas<BR>5323 Harry Hines Blvd.<BR>Dallas, TX 75390<BR>Office: (214) 645-6384<BR>Fax: (214) 645-5383<BR><BR><BR><BR><BR>_______________________________________________<BR>RASMB mailing list<BR>RASMB@rasmb.bbri.org<BR><A href="http://rasmb.bbri.org/mailman/listinfo/rasmb">http://rasmb.bbri.org/mailman/listinfo/rasmb</A><BR></DIV></BODY></HTML>