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Hi Christopher,<br><br>
just as a reminder, as you noted, SEDFIT consistently calculates the
distributions in experimental s-values. There are utility functions
to convert it to s20w on the request of the user (for example the Mw
button appearing over each peak after automatic integration will prompt a
report among other things like mass and shape estimates also the s20w
value). <br><br>
The reason why it does experimental s-values is that in most cases with
c(s) you will be able to resolve more than one species. Since the
transformation to s20w includes a buoyancy correction term, there would
really need to be more than one vbar value to do the correction (... one
for each peak...) SEDFIT leaves it up to you to identify which c(s) peak
you're interested in and assign the vbar value accordingly.
Otherwise if you assume a single vbar value for all species in the
distribution, some of the s-values may be off. That would be a
waste since the experimental s-values are the most precisely determined
quantities (after the concentrations).<br><br>
Peter<br><br>
<br>
At 12:29 PM 3/20/2008, Chin, Christopher C. wrote:<br>
<blockquote type=cite class=cite cite="">
<font face="arial" size=2 color="#000080">Hello Marina,<br>
<br>
As I try to clean up my old e-mail and notice that I miss this discussion
about two weeks ago. I remember I did an experiment when Sedfit
was release. The reason for doing this experiment is when I analyzed the
sample with glycerol presence in the buffer, the S value is always
smaller than when I analysis the data using DCDT +, even when I put in
the correct density and viscosity value. So I derived an experiment
using CRP protein (a dimmer protein with molecular weight about 47KDa) in
the presence of different concentration of glycerol. I am sending this in
the attachment. If I use the correction constant, then your protein of
</font><font face="arial" size=2 color="#FF0000">6.23S
</font><font face="arial" size=2>should really be 1.21X
6.23=</font><font face="arial" size=2 color="#FF00FF">7.54
S</font><font face="arial" size=2 color="#000080">. This will reconfirm
it is a trimmer but will not change the trimmer to hexamer as you
expected. I hope this information is useful for you.<br>
<br>
All the best,<br>
<br>
Christopher Chin<br>
<br>
<br>
</font><font face="tahoma" size=2>-----Original Message-----<br>
<b>From:</b> rasmb-bounces@rasmb.bbri.org
[<a href="mailto:rasmb-bounces@rasmb.bbri.org" eudora="autourl">
mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Thomas
Jowitt<br>
<b>Sent:</b> Monday, March 03, 2008 4:39 AM<br>
<b>To:</b> thomas.a.jowitt@manchester.ac.uk; Fasolini, Marina
[Nervianoms]; rasmb@rasmb.bbri.org<br>
<b>Subject:</b> RE: [RASMB] problem with fitting<br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2 color="#000080">Hi Marina<br>
<br>
Just a follow up to my previous reply. I notice you are running the
sample in 5% glycerol? The viscosity of this alone is 1.127E-2, not what
you have mentioned in your mail. If this is the case then this would
alter the S20,w of your protein substantially. <br>
<br>
Thanks<br>
<br>
Tom<br>
<br>
<hr>
<div align="center"></font></div>
<font face="tahoma" size=2><b>From:</b> rasmb-bounces@rasmb.bbri.org
[<a href="mailto:rasmb-bounces@rasmb.bbri.org" eudora="autourl">
mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Thomas
Jowitt<br>
<b>Sent:</b> 03 March 2008 10:17<br>
<b>To:</b> Fasolini, Marina [Nervianoms]; rasmb@rasmb.bbri.org<br>
<b>Subject:</b> RE: [RASMB] problem with fitting<br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2 color="#000080">Dear Marina<br>
<br>
First of all, unless your gel filtration is linked to a light scattering
module to estimate the absolute molecular mass, then I wouldn’t trust the
estimated molecular weight from a calibrated column. A sedimentation
value of 6.23 is most likely to be around 150kDa unless the molecule is
substantially elongated, as a hexameric molecule of this size and
sedimentation value would have a frictional ratio of around 2.5
(certainly not impossible). <br>
<br>
The two experiments that I would perform to answer these questions are,
firstly an equilibrium experiment to establish the correct molecular
weight, done with at-least three concentrations to establish if
oligomerisation is concentration dependant. Secondly, I would run the
crystal structure through a modeling program such as SOMO to generate a
bead model, or hydropro to create a shell around the crystal structure.
Either of these would give you a decent estimate of the sedimentation
value expected for a rigid molecule of that size. <br>
<br>
Thanks<br>
<br>
Tom Jowitt<br>
<br>
<br>
<hr>
<div align="center"></font></div>
<font face="tahoma" size=2><b>From:</b> rasmb-bounces@rasmb.bbri.org
[<a href="mailto:rasmb-bounces@rasmb.bbri.org" eudora="autourl">
mailto:rasmb-bounces@rasmb.bbri.org</a>] <b>On Behalf Of </b>Fasolini,
Marina [Nervianoms]<br>
<b>Sent:</b> 03 March 2008 09:10<br>
<b>To:</b> rasmb@rasmb.bbri.org<br>
<b>Subject:</b> [RASMB] problem with fitting<br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2>Dear AUC users,<br>
I have a problem in the fitting of some data and I would like to have
your opinion.<br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2>I want to control the sedimentation of a
protein in order to define the state of oligomerization. It is published
as forming an hexameric homo oligomer. From the crystal structure,
it is a wheel like structure of 160 angstrom in diameter, with a central
role of 15 angstrom in diameter. The thickness is 20-40 angstrom. How can
I expect it to sediment? How can I improve my parameters or model in
order to improve the fitting?<br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2>The protein is 50KDa but in gel
filtration it cames out as hexamer (300 KDa). In my sedimentation
experiment I see one peak of 6.23S which corresponds to 140KDa.
</font><font face="arial" size=2 color="#0000FF">Fitting of the
sedimentation data was done with SedFit.
</font><font face="arial" size=2>Do you think I can say that it is a
trimer? I would expect it as an hexamer.<br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2>I hope
someo</font><font face="arial" size=2 color="#0000FF">ne
can</font><font face="arial" size=2> help
me</font><font face="arial" size=2 color="#0000FF">. <br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2>the conditions are : <br>
buffer 20mM Tris pH7, 150mM NaCl, 1mM DTT, 5%gly <br>
Vbar 0.7397 <br>
Viscosity 1.02530 <br>
Density 0.01183 <br>
</font><font face="Times New Roman, Times"> <br>
</font><font face="arial" size=2>Thanks a lot<br>
Marina <br>
</font><font face="Times New Roman, Times"> <br>
<br>
</font><font face="Times New Roman, Times" size=2> <br>
</font><font face="arial" size=2>MARINA FASOLINI<br>
Structural Chemistry <br>
<a href="http://www.nervianoms.com/">Nerviano Medical Sciences</a><br>
Viale Pasteur 10<br>
20014 Nerviano - Milano<br>
<a href="mailto:marina.fasolini@nervianoms.com">
marina.fasolini@nervianoms.com</a><br>
Tel. +390331581462<br>
Fax. +390331581360<br>
</font><font face="Times New Roman, Times"> <br>
</font><br>
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