[RASMB] Sedimentation equilibrium question
Arthur Rowe
arthur.rowe at nottingham.ac.uk
Thu Mar 29 08:28:25 PDT 2007
Greetings, everyone
Jo makes a point (below) which is in fact a general one. Assuming your
software allows it (and I know that Jo and I both use pro Fit, which makes
it easy) then every time you fix a parameter in a fit you should at least
give thought to constraining that value within error bounds. Unless it is
something like a radial value, where error can be taken as nil.
For a protein, the vbar value is indeed critical in evaluating sigma. Errors
in solvent density you can pretty much forget about, so final error in the
(1 - vbar* rho) term ought to be about 3 times greater than the uncertainty
in (usually computed via SEDNTERP) vbar value you use. I would reckon that
±1% in sigma would be OK for most cases.
That may sound a bit optimistic. But we need to bear in mind that if you do
have a system of some polydispersity, then the sigma value tends to be
significantly correlated with the (floated) baseline (E) value. So - whilst
I would 'give thought' to floating, I am not sure it would always be very
positive thought. There is a danger that shoving in one extra parameter into
a fit one may merely permit that fit to take advantage of the elbow room
given by data error to seek out an optimal chi-squared/whatever.
Arthur
Dear Sarah,
If I may chip in again, Arthur is basically correct - one usually knows
the actual monomer mass very accurately from sequence - but there is
still the problem of the v-bar to use for the residual mass correcting
for Archimedian buoyancy [i.e. M(1 - v-bar*rho)], which is why it is
usual to float the mass, at least within bounds.
One can also get interesting problems, such as one protein I worked on
which appeared as a dimerising, non-ideal dimer - which would never
dissociate (under non-denaturing conditions) to monomer. It was a
couple of years later that my colleagues solved the crystal structure
and showed that it was a coiled-coil dimer, which would snap together on
synthesis and not dissociate again!
Jo
Arthur Rowe wrote:
> Hi Sarah
>
> There are lots of good suggestion coming your way - so I will not
> duplicate. But one little point does occur to me. You sound to be
> floating the (reduced) molecular weight of your protein. Assuming that
> what you have is a chemically pure system (i.e. there is only one
> covalently bound entity present (you say it is 84 kDa, purified) then
> you should be /fixing/ this parameter for the monomeric species,
> whatever mode of analysis you use.
>
> Oh, and one little thing more. There are dangers in the "try every
> model you can think of, select the one which has best
> residuals/whatever as being _correct_" approach. It has long been
> appreciated that simple theory shows that if you keep on doing this,
> then you will always, eventually, come up with something that looks
> good. Doesn't mean it's right, though. The famous example of this is
> the search (a few years back) for ways of predicting earthquakes -
> which eventually came up* with a statistically good correlation
> between occurrence of terrestrial earthquakes and the phases of a moon
> of the planet Uranus!
>
> Kind regards
>
> Arthur
> *the authors /did/ report this with tongue firmly in cheek, I should add.
> -
> *************************
> Arthur Rowe
> Lab at Sutton Bonington
> tel: +44 115 951 6156
> fax: +44 115 951 6157
> *************************
>
>
> *From: *Tom Laue <tml at cisunix.unh.edu>
> *Organization: *CAMIS-UNH
> *Reply-To: *Tom.Laue at unh.edu
> *Date: *Thu, 29 Mar 2007 09:06:47 -0400
> *To: *Sarah Siegel <ssiegel at scripps.edu>
> *Cc: *rasmb at server1.bbri.org
> *Subject: *Re: [RASMB] Sedimentation equilibrium question
>
>
> Hi Sarah-
> Have you done sedimentation velocity analysis of your samples?
> What does
> it show with respect to the concentration dependence of the g(s)
> patterns? This sort of analysis will tell you whether it is
> worthwhile
> doing equilibrium sedimentation by: 1) showing whether there is
> aggregate, 2) showing if there is significant mass action
> association/dissociation in the concentration range where you are
> working and 3) making sure there are no smaller species.
> With respect to the equilibrium analysis- fit the individual data
> sets
> first to a simple model (average molar masses as a function of
> concentration).Essentially doing what Jo and Ariel suggested.
> There are
> several programs to help with this: Sedanal has Biospin built into
> it,
> MStar, Segal. The trick with any of these analyses is making sure the
> signal is equal to the concentration, and it is the presence of any
> baseline offset (i.e. zero signal does not equal zero concentration)
> that is the usual concern. For absorbance data, the baseline offset
> usually is small and can be determined experimentally by pelleting
> your
> material (or at least making sure some data are acquired at a rotor
> speed where the meniscus region is entirely depleted of protein).
> Interference optics are tougher to deal with since the baseline
> offset
> may change with rotor speed (due to window stresses). You may use
> HeteroAnalysis, fitting to an ideal single component and fit for the
> baseline offset. In this latter case, M will be between the weight
> and
> z- average, typically closer to the latter.
> See if the individual data sets reveal systematic behavior with
> respect
> to concentration and rotor speed. If the M increases with
> concentration
> (I would suggest acquiring data over a wider initial concentration
> range), then you have a mass action association, plus you have
> some idea
> (within a decade) of the monomer concentration range where the mass
> action association is important to protein behavior. If M decreases
> significantly with increasing rotor speed, you probably have
> irreversible aggregate (but you would know this clearly from velocity
> analysis).
> Best wishes,
> Tom
>
> Sarah Siegel wrote:
> > Hi Everyone,
> >
> > I am a beginner in the world of AUC and I'm having a lot of trouble
> > fitting sedimentation equilibrium data. I was hoping someone could
> > help me figure out if my problem is in the way I'm fitting or
> the way
> > I set up my experiment. I did attend last spring's AUC workshop at
> > UConn, but this is my first time really running an equilibrium
> > experiment. The system I have is an 84 kDa purified protein
> that may
> > be in equilibrium with dimer, possibly also some larger species.
> I am
> > trying to get an idea of what the dominant species present are.
> I ran
> > two samples, at 1.0 and 0.5 mg/mL, collecting interference data
> at a
> > range of speeds from 8,000 rpm to 20,000 rpm. I watched the
> approach
> > to equilibrium using Match in HeteroAnalysis. I have been using
> > HeteroAnalysis to try to fit the data globally. I have tried
> fitting
> > to several models, including ideal and nonideal systems,
> > monomer-dimer, monomer-trimer, monomer-tetramer, and several three
> > species equilibria. So far the best fit I have has rms
> deviations of
> > 0.027 using a nonideal model, however this fit also tells me the
> > molecular weight of the protein is 138 kDa, which is larger than
> > monomer and smaller than dimer, and the residuals do not have a
> random
> > pattern. Does anyone have any ideas about another way I should be
> > fitting this, or other data I should collect? Thanks for your help!
> >
> > Sarah
> >
> > Sarah Siegel
> > Graduate Student
> > The Scripps Research Institute
> > La Jolla, CA
> > _______________________________________________
> > RASMB mailing list
> > RASMB at rasmb.bbri.org
> > http://rasmb.bbri.org/mailman/listinfo/rasmb
> >
>
> --
> Department of Biochemistry and Molecular Biology
> University of New Hampshire
> Durham, NH 03824-3544
> Phone: 603-862-2459
> FAX: 603-862-0031
> E-mail: Tom.Laue at unh.edu
> www.bitc.unh.edu
> www.camis.unh.edu
>
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