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<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Hello </span></font><font size=2
color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
color:navy'>Marina</span></font><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>,</span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>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 </span></font><font size=2 color=red face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:red'>6.23S </span></font><font
size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'>should
really be 1.21X 6.23=<font color=fuchsia><span style='color:fuchsia'>7.54 S</span></font><font
color=navy><span style='color:navy'>. 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.</span></font></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>All the best,</span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Christopher Chin</span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Tahoma><span
style='font-size:10.0pt;font-family:Tahoma'>-----Original Message-----<br>
<b><span style='font-weight:bold'>From:</span></b> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <b><span style='font-weight:bold'>On
Behalf Of </span></b>Thomas Jowitt<br>
<b><span style='font-weight:bold'>Sent:</span></b> </span></font><font size=2 face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>Monday, March
03, 2008</span></font><font size=2 face=Tahoma><span style='font-size:10.0pt;
font-family:Tahoma'> </span></font><font size=2 face=Tahoma><span
style='font-size:10.0pt;font-family:Tahoma'>4:39 AM</span></font><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'><br>
<b><span style='font-weight:bold'>To:</span></b>
thomas.a.jowitt@manchester.ac.uk; Fasolini, Marina [Nervianoms];
rasmb@rasmb.bbri.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> RE: [RASMB] problem with
fitting</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>Hi Marina</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>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. </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>Thanks</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>Tom</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<div>
<div class=MsoNormal align=center style='margin-left:.5in;text-align:center'><font
size=3 face="Times New Roman"><span style='font-size:12.0pt'>
<hr size=2 width="100%" align=center>
</span></font></div>
<p class=MsoNormal style='margin-left:.5in'><b><font size=2 face=Tahoma><span
style='font-size:10.0pt;font-family:Tahoma;font-weight:bold'>From:</span></font></b><font
size=2 face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <b><span
style='font-weight:bold'>On Behalf Of </span></b>Thomas Jowitt<br>
<b><span style='font-weight:bold'>Sent:</span></b> 03 March 2008 10:17<br>
<b><span style='font-weight:bold'>To:</span></b> Fasolini, Marina [Nervianoms];
rasmb@rasmb.bbri.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> RE: [RASMB] problem with
fitting</span></font></p>
</div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>Dear Marina</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>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). </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>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. </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>Thanks</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>Tom Jowitt</span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in'><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'> </span></font></p>
<div>
<div class=MsoNormal align=center style='margin-left:.5in;text-align:center'><font
size=3 face="Times New Roman"><span style='font-size:12.0pt'>
<hr size=2 width="100%" align=center>
</span></font></div>
<p class=MsoNormal style='margin-left:.5in'><b><font size=2 face=Tahoma><span
style='font-size:10.0pt;font-family:Tahoma;font-weight:bold'>From:</span></font></b><font
size=2 face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <b><span style='font-weight:bold'>On
Behalf Of </span></b>Fasolini, Marina [Nervianoms]<br>
<b><span style='font-weight:bold'>Sent:</span></b> 03 March 2008 09:10<br>
<b><span style='font-weight:bold'>To:</span></b> rasmb@rasmb.bbri.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> [RASMB] problem with
fitting</span></font></p>
</div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Dear AUC users,</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>I have a problem in the fitting of
some data and I would like to have your opinion.</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>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?</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>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 color=blue><span style='color:blue'>Fitting
of the sedimentation data was done with SedFit. </span></font>Do you think
I can say that it is a trimer? I would expect it as an hexamer.</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>I hope someo<font color=blue><span
style='color:blue'>ne can</span></font> help me<font color=blue><span
style='color:blue'>. </span></font></span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>the conditions are : </span></font></p>
</div>
<div>
<div u1:shape="_x0000_s2050">
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>buffer 20mM Tris pH7, 150mM NaCl,
1mM DTT, 5%gly </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Vbar 0.7397 </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Viscosity 1.02530 </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Density 0.01183 </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Thanks a lot</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Marina </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'> </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face="Times New Roman"><span
style='font-size:10.0pt'> </span></font></p>
</div>
</div>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>MARINA FASOLINI</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Structural Chemistry </span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'><a href="http://www.nervianoms.com/">Nerviano
Medical Sciences</a></span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Viale Pasteur 10</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>20014 Nerviano - Milano</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'><a
href="mailto:marina.fasolini@nervianoms.com">marina.fasolini@nervianoms.com</a></span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Tel. +390331581462</span></font></p>
</div>
<div>
<p class=MsoNormal style='margin-left:.5in'><font size=2 face=Arial><span
style='font-size:10.0pt;font-family:Arial'>Fax. +390331581360</span></font></p>
</div>
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style='font-size:12.0pt'> </span></font></p>
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