[RASMB] error estimates and F-statistics in Sedphat

[John Philo]

Mark,
 
Given that the fit is so bad I'm not sure it makes any sense to try to get a
confidence interval. The model is clearly not even coming close to
representing the data, so what does this association constant mean? 
 
I agree fully with Jack that you cannot expect the peaks from c(s) analysis
to give you the monomer and dimer sedimentation coefficients, and that you
really want to do a dilution series over a big concentration range. For
absorbance data that usually means either using multiple wavelengths (in
different runs) or using the 3 mm centerpieces to help cover a big range
while keeping the OD <=1 for the high end. Then you can either fit the
weight-average sedimentation coefficient versus concentration to a model, or
do global whole boundary analysis with SEDPHAT or SEDANAL (with the
sedimentation coefficients as fitting variables).
 
For this or any other experiment I also recommend that you fill you cells
more completely to get a better separation. Try to get the meniscus at
5.9-5.95 cm. 
 
John

  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Mark Agacan
Sent: Tuesday, April 26, 2011 8:36 AM
To: RASMB at rasmb.bbri.org
Subject: Re: [RASMB] error estimates and F-statistics in Sedphat


Thanks Patrick and Chad for the information about error estimates from
F-statistics.
 
My problem now is that the apparent errors for the binding constants derived
from the analysis of F-statistic appear to be very large.  When I make a
change to e.g. log10Ka12, by any value from 0.1 - 3.0, there is no change
(or only a very small change) in chi squared, so I can never reach the
critical chi squared.  
 
Each dataset looks good and gives low rmsd values when processing with the
c(s) model in sedfit.  I'm using the monomer-dimer self-association model in
sedphat, with the values for S1 and S2 taken from c(s) analysis.  I have
tried to fit to other models with only worse results.
 
The fit looks very poor (see screenshots attached).  I don't think I need to
repeat the experiment because the data looks good, at least to my eye, but
the fit to the monomer-dimer model seems very bad.  
 
Any further suggestions or advice would be very welcome.
 
Many Thanks,
 
Mark
 

 
 
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Dr Mark Agacan, Scientific Officer for the Division of Biological Chemistry
and Drug Discovery,
Wellcome Trust Biocentre, College of Life Sciences, University of Dundee,
Dundee, DD1 5EH 
Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 
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>>> Chad Brautigam <Chad.Brautigam at UTSouthwestern.edu> 4/23/2011 15:02 >>>

Hi, Mark,

The F-statistics in SEDPHAT are meant to allow you to construct a
"do-it-yourself" error interval.  Check out the statistics help page.  Under
the heading "Error Analysis" there is a procedure for how to use the output
"critical chi-square" from the Statistics Menu to find an error interval.

Cheers,
Chad


  _____  

From: rasmb-bounces at rasmb.bbri.org [rasmb-bounces at rasmb.bbri.org] on behalf
of Mark Agacan [M.Agacan at dundee.ac.uk]
Sent: Friday, April 22, 2011 6:36 AM
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] error estimates and F-statistics in Sedphat


Hello,
 
I'm calculating errors in the binding constants in sedphat as instructed in
the statistics help page, but I don't appear to get an output for the
F-statistics.  Are the F-stats output only as (changes in) chi-squared
values? e.g. the error associated with the binding constants is clearly
given in the covariance matrix.
 
Best Wishes,
 
Mark
 
 
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Dr Mark Agacan, Scientific Officer for the Division of Biological Chemistry
and Drug Discovery,
Wellcome Trust Biocentre, College of Life Sciences, University of Dundee,
Dundee, DD1 5EH 
Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 
 
************************************************************ 
Please consider the environment. Do you really need to print this email? 


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