[RASMB] problem with fitting

Thomas Jowitt thomas.a.jowitt at manchester.ac.uk
Mon Mar 3 02:39:22 PST 2008


ProgId Word.Document Generator Microsoft Word 11 Originator Microsoft Word 11 Hi Marina

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 

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Thomas Jowitt Sent: 03 March 2008 10:17 To: Fasolini, Marina [Nervianoms]; rasmb at rasmb.bbri.org Subject: RE: [RASMB] problem with fitting

Dear Marina

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). 

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. 

Thanks

Tom Jowitt


From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Fasolini, Marina [Nervianoms] Sent: 03 March 2008 09:10 To: rasmb at rasmb.bbri.org Subject: [RASMB] problem with fitting

Dear AUC users,
I have a problem in the fitting of some data and I would like to have your opinion.

I want to control the sedimentation ofaprotein in order to define the state of oligomerization.It is published as forming anhexameric homo oligomer.From the crystal structure,it is a wheel like structure of 160angstrom 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?

The protein is 50KDabut ingel filtration it cames out ashexamer (300 KDa).In my sedimentation experiment I seeonepeak of6.23S which corresponds to 140KDa.Fitting of the sedimentation data was done with SedFit.Do you think I can say that it is a trimer? I would expect it as anhexamer.

I hopesomeonecan help me.

the conditions are :
buffer 20mM Tris pH7, 150mM NaCl, 1mM DTT, 5%gly 
Vbar 0.7397 
Viscosity 1.02530 
Density 0.01183 

Thanks a lot
Marina



MARINA FASOLINI
Structural Chemistry 
Nerviano Medical Sciences
Viale Pasteur 10
20014 Nerviano - Milano
marina.fasolini at nervianoms.com
Tel. +390331581462
Fax. +390331581360
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