[RASMB] Working with glycerol solutions

[Peter Schuck]

Hi Chris,

In the nice experiments shown in your attached document you made a very 
important observation, which I would like to highlight in the discussion 
about working in glycerol:  The experimental s-value decreases as the 
glycerol concentration increases. 

The point is that SEDFIT does not make any buffer corrections on the 
s-values (with the exceptions of very few specialized models where a 
notice to that effect will pop up).  This is despite the fact that you 
enter vbar and rho in the parameter box for the distribution analysis.  
Therefore, all values determined from SEDFIT will be treated as 
experimental values, and should be corrected with the appropriate 
correction factor.  The correction factor can be determined, as you did, 
using SEDNTERP, or using a calculator function in the Options menu of 
SEDFIT. 

[The benefit from entering the correct viscosity, buffer density, and 
vbar in the c(s) model of SEDFIT is that the frictional ratios will be 
scaled to the familiar units, and that c(M) can give you mass values on 
the correct scale, and if you're not interested in that, you can leave 
this at the default values.]

Admittedly, that seems counter-intuitive, but is this way for reasons of 
consistency between the different models, and to separate issues of 
experimental conditions from the actual data analysis.  Also, for a 
proper correction of the s-values, the vbar of the sedimenting particles 
is required, which sometimes we may not know or may be different for 
different species within a distribution of molecules.  To defer all 
these issues, this correction is left up to the user to do.

This is different from SEDPHAT, which is does the buffer correction when 
supplied with the correct density and viscosity values.  The reason why 
we cannot avoid buffer correction here is that this is a platform for 
global analysis.  (For example, you should be able to load all data sets 
at once, and see if they fit a single s-value when you enter the proper 
density and viscosity values for each experiment.)  In order to enable 
global analysis, one needs to correct the values to a reference 
condition (i.e. water, 20C). 

Best,
Peter



Chin, Christopher wrote:

> Dear Jo,
>  
> I have done experiment using a pure protein in various concentration 
> of glycerol (0-10%) as a project to familiar myself with the SEDFIT 
> software a while back.  I also used the same protein and watch how 
> dimer dissociated in the presence of GuHCl (0-5M) analyzed with the 
> same software. I will share these results if anyone is interested.
>  
> Sucequently, I was interested in how to choose scan, the # of scan 
> used to get the best fit and analyzed the raw data with both SEDFIT 
> and DCDT+ and compare these results.  I also did some experiments, 
> using a dimer protein and observe what happen in the presence of 
> various concentration of denaturant(GuHCl), as mentioned above, 
> but analyzed the raw data with both SEDFIT and DCDT+ and compare the 
> results.  I can also share these results in case anybody is interested.
>  
> I did these experiments as an in house projects, Just to familiar 
> myself, as an experimentalist, not  theoretician, to observe the pro 
> and con of using these two softwares. Enough been said.
>  
> Regards,
>  
> Chris
>
>  
>
>  
>
>  
>
> ------------------------------------------------------------
>
> Christopher Chin
>
> Manager, Macromolecular Assembly Core
>
> Sealy Center for Structural Biology and 
> Molecular Biophysics
>
> Department of Biochemistry & Molecular Biology
>
> 5134 MRB. rt1055
>
> UTMB, Galveston, TX  cchin at utmb.edu <mailto:cchin at utmb.edu>,
>
> 409-772-1693, efax 630-604-3416
>
> -------------------------------------------------------------
>
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