[RASMB] Re: RASMB digest, Vol 1 #319 - 5 msgs

Thu Jul 15 11:20:01 PDT 2004

Dear RASMB,

I attached a pdf file (distribution.pdf) containing c(s) distribution plot
at high and low protein conecentration. The parameters are included, also
the rmsd, f/f0, residual bitmap. p = 0.68 and the resolution is 250 (smin to
smax is 0.1 to 25). Thanks everyone's comments and suggestion.

Chi-Yuan Chou
PhD student, the Institutes of Life sciences, National Defense Medical
Center, Taipei, Taiwan
e-mail: r6243023 at yahoo.com.tw
Lab homepage: http://www.enzkin.org/

----- Original Message ----- 
From: "John Correia" <jcorreia at biochem.umsmed.edu>
To: <ls890067 at ndmctsgh.edu.tw>; <rasmb at server1.bbri.org>
Sent: Thursday, July 15, 2004 6:45 AM
Subject: Re: [RASMB] Re: RASMB digest, Vol 1 #319 - 5 msgs

> Isolating the main peak, reconcentrating the sample, and generating the
> same distribution suggests reversibility, the classic test, although its
> not clear what a 10 peak pattern means?  Discrete peaks suggest
> irreversible aggregates.  Is there reducing agent in the buffer?  Cys in
> the protein?
>
> What optical system and how good are the fits, rms values?   c(s) can
> get more peaky with very low noise levels & apparently low p values - I
> personally only do .95.  I always check c(s) distributions with g(s),
> although for a broad distribution g(s) may be hard to apply.
> Alternatively what do the Ls-g(s) distributions look like?  Ultimately I
> plot c(s) and g(s) as a function of concentration to develop hypotheses
> about the data.  The shape of the conc dependence of the boundaries is
> often informative, although I would never fit a c(s) or Ls-g(s)
> distribution shape to extract molecular information.  Maybe a g(s) since
> if properly done its the derivative of the boundary.
>
> Then I go to direct boundary fitting, individually & globally - in my
> case Sedanal but sedfit/sedphat can work depending upon the model.
> Years ago (the 70's) there was a lively, but non email (didn't exist),
> discussion about fitting raw data vs smoothed data or extracted moments
> - many methods give similar answers, but if possible always fit the raw
> data directly.
>
> So if your system is a broad but reversible distribution what are the
> options?  Indefinite?  You are describing big shifts?  You need to
> establish endpoints, the s value of the monomer and the endpoint of the
> association, if there is one?  Integrate the entire distribution and
> plot weight average S vs concentration.  Remember the behavior of a
> simple titration experiment, you need at least two orders of magnitude
> to go from 10% to 90% saturation.  Plot the data vs log conc.  Does it
> look like a binding curve and does it saturate?  To go higher in c try
> the 0.3 mm path centerpieces, you can gain a factor of 5 in
> concentration.  Try 230 nm or interference to go lower, or can you
> estiamte s1 from the other isoform's data?
>
> Without seeing the data or the actual distributions it is hard to know!
>
>
>
> -------------------------------------------------------------------
>  Dr. John J. "Jack" Correia
>  Department of Biochemistry
>  University of Mississippi Medical Center
>  2500 North State Street
>  Jackson, MS  39216
>  (601) 984-1522
>  fax (601) 984-1501
>  email address: jcorreia at biochem.umsmed.edu
>  homepage location: http://biochemistry.umc.edu/correia.html
>  dept homepage location:    http://biochemistry.umc.edu/
> -------------------------------------------------------------------
>
>
>
> >>> "medakachou" <ls890067 at ndmctsgh.edu.tw> 07/13/04 11:38 PM >>>
> --------------------------------------------------------------------------
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>
> > Date: Tue, 13 Jul 2004 10:59:00 -0500
> > From: "John Correia" <jcorreia at biochem.umsmed.edu><~!B*+R^&>> To:
> <jphilo at mailway.com>, <r6243023 at ms48.hinet.net>,<~!B*+R^&>>
> <arthur.rowe at nottingham.ac.uk>, <rasmb at server1.bbri.org><~!B*+R^&>>
> Subject: Re: [RASMB] difference of p = 0.95, 0.68 and 0.55, the
> > confidencelevel in the sedfit c(s) distributi
> >
> > This is a MIME message. If you are reading this text, you may want to
> > consider changing to a mail reader or gateway that understands how to
> > properly handle MIME multipart messages.
> >
> > --=__Part0E2FB7D4.3__=
> > Content-Type: text/plain; charset=US-ASCII
> > Content-Transfer-Encoding: 7bit
> >
> > One problem I have with these discussions is they are method of
> analysis
> > focused and until Arthur's illustion to "know your system" not focused
> > on the molecules, the mechanism, the interactions.  Were we just
> talking
> > about one run and one concentration or a series of conectrations?  Why
> > do you want to integrate?  Are the "peak" positions constant or do
> they
> > change with concentration?  Is this in fact an impure system of
> > nointeracting species or aggregates, or are these aggregates of a
> single
> > component?  Is there any reversible interaction going on?
> >
> > The goal is to describe your system in molecular and mechanistic terms
> > and then fit the data individually & globally to that model to prove
> the
> > hypothesized mechanism.  Statistics, assumptions, simulations are all
> > important.  Now what is going on in your system?
>
> Dear John,
>
>     Yes, I should give more information about my case. I expressed and
> purified a 34 kDa protein and it's N-terminal or C-terminal truncated
> fragments by E. coli expression system. The protein purity is > 99% by
> SDS-PAGE.  This protein has two isoforms. I study them in three
> different
> concentration: 0.15, 0.50 and 1.00 mg/ml in PBS (pH7.3). By using
> sedimentation velocity and c(s) distribution analysis, I found one
> isoform's
> N-terminal truncated fragments showed a 10 "peaks" pattern whose s is
> from 3
> to 23 at 1.00 mg/ml. While at 0.15 mg/ml, only 5 peaks were found at s =
> 3
> to 12. It means it should be a single component aggregation and is
> concentration-dependent. The other isoform didnot show this
> characteristics.
> I want to give my paper some quantitative data about this difference, so
> I
> chose "Origin peak fitting module" and analyzed the pattern of gaussian
> peaks. The reviewer thought it is overinterpretation (about the
> fused-"peaks") and suggested me lowering the cinfidence level to p =
> 0.7.
> I've tried and found the resolution is better (every "peaks" is still
> existed). These two days I've tried Jack Lebowitz's comment and gained
> some
> quantitative data. I'm going to compare them and hope it can make my
> paper
> more quantitative sound.
>     By the way, while I isolated the major "peak" species by using
> gel-filtration chromatography (S-300 column) and concentrate them (I
> need
> higher concentration), It just change back to the same "multi-peaks"
> situation (by sedimentation velocity). I think it's not a
> non-interacting
> but a associating system, right? Thanks your help.
>
> Chi-Yuan Chou
> PhD student, the Institutes of Life sciences, National Defense Medical
> Center, Taipei, Taiwan
> e-mail: r6243023 at yahoo.com.tw
>
>
>
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