Fwd: RE: [RASMB] Sedimentation Velocity Experiments that are hard to fit

[David Hayes]

Hi John,

Thank you for your comments.

I think it is OK to say that the data is absorbance and that it looks like 
a picture from a Beckman training manual.  I was a little surprised they 
were interested enough in the truth to let me describe the problem I was 
having on RASMB in this general way to get community input, but I don't 
want to push things by divulging any details:  I actually do not know 
myself what the buffer is.  I mention non-ideality because the darn scans 
just don't fit as a whole but each one looks fine by itself.
I might be more interested in getting a good fit than the people who own 
the data:  but it really bugs me that something that looks so simple 
doesn't fit the models I tried in Sedfit and Sedanal.

Thanks

David Hayes



>David,
>
>It is hard to comment without knowing more about the experiment or seeing 
>the data. Can you at least tell us whether you are talking about 
>absorbance or interference data?
>
>You mention that you have considered non-ideality, which suggests that 
>this could be an experiment at high protein concentration. Is that 
>correct? If so, then part of the problem might be optical distortions due 
>to refraction by the boundary---these are always worst early in the run, 
>and go away as diffusion makes the gradient less steep. That problem can 
>be significantly reduced by using 3 mm centerpieces.
>
>John
>-----Original Message-----
>From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] 
>On Behalf Of David Hayes
>Sent: Friday, March 24, 2006 12:41 PM
>To: RASMB
>Subject: [RASMB] Sedimentation Velocity Experiments that are hard to fit
>
>Hi to all experienced Ultracentrifuge Scientists,
>
>I have some data (repeated experiments) from sample A buffer 1 (which may 
>or may not have been produced on the planet earth -- that is all I can say 
>about it)
>
>There are two puzzling aspects to this data.
>First, I am not getting good repeatability in the amount of aggregate.
>Second, by eye, the raw data looks like a single species (which it should 
>be) with a small amount of higher weight aggregate (not surprising), but 
>the entire data set never fits well no matter what I try.
>Thinking that the repeatability problem might be an artifact of my 
>inexperience using Sedfit I tried many different strategies:  including 
>floating or not floating just about every parameter and also using the 
>nifty "experimental initial distribution" option of Sedfit.
>   Fitting to the whole data set, I got a fit with a single large peak and 
> two very small aggregate bumps which all together integrate to about 4% 
> of the total.  The rmsd was a bit large at 0.01 and the residuals were 
> systematic.  Early scans were fit very poorly and later scans fit 
> somewhat poorly with scans in the middle fit pretty well.  Later using 
> Sedfit with the single species model and only the later half of the scans 
> and floating a lot of things and turning off the hat function in Sedfit 
> brought me down to a comparable fit with a rmsd of .006, but the 
> residuals were still systematic and the molecular weight was off more 
> than what I expected for having 4% aggregate.
>Then I did something that Peter Shuck thinks is a bit strange, but I think 
>it helped me understand the data a bit more.  I fit pairs of scans with 
>c(s) taking scans at different times paired with the last 
>scan.  Essentially then, I was fitting each scan individually:  using the 
>last scan in ever pair was needed to help Sedfit know the baseline.  Peter 
>reminded me that by fitting each scan, I lose the ability to distinguish 
>heterogeneity from diffusion.  But in this case, all the evidence points 
>to a single species, so I did not think heterogeneity (beyond the measured 
>4% aggregation) was significant.  The peak S value stays constant all the 
>way down the cell.  Each individual scan fit this way fit very well 
>without systematic residuals and a rmsd of about .005.  What changed was 
>that early scans fit to an f/f0 of 1.06 while by the later scans f/f0 fit 
>to 1.5.  This means that the early scans are broader than they should be 
>for a reasonable f/f0 and that the later scans seem to be narrower and 
>diffusing less.
>I repeated this with Sedanal dcdt based curve fitting (here I took scans 
>by twos going down the cell and fit to a single species ignoring the 4% 
>aggregate) and found a similar trend:  the fitted molecular weight went 
>from 95,000 to 225,000 and then settled down to about 160,000.  If there 
>were heterogeneity, the scans would fit the opposite way to a one species 
>model, the f/f0 would go down, or the weight would go up later in the run 
>as the species sedimented apart.
>
>I had some interference data of Tropomyosin that I subjected to a similar 
>type of analysis with Sedanal, fitting the whole data set and then fitting 
>scans by sets of 10 for the different times in the experiment (because of 
>TI, RI noise fitting, it is not possible to fit small sets of scans with 
>Sedfit using interference data; however, a Sedfit fit to the whole Tm data 
>set showed no systematic variation of the residuals dependent on scan 
>number).  There was no scan number dependent trend in molecular weight 
>with this molecule, though the variability in fitted molecular weight was 
>surprising (weights from different sets of 10 varied from 60,000 to 80,000 
>randomly).
>
>Seeing this I tried a Sedfit single species and Sedanal single species 
>fits with non-ideality parameters, but floating these parameters made no 
>substantial difference.
>
>I don't know if anyone has run into this type of problem, but I am out of 
>ideas what to try next.  I am being a bit stubborn wanting a good fit to 
>the data:  the S value of the main peak is reproducible and the data 
>contains quite a bit of information, but I can't figure out exactly what 
>is going on:
>
>Is this just non-ideality that I am not fitting properly or maybe that is 
>not modeled well?
>Is the culprit convection?  Thoughts on convection in the next message.
>
>Cheers
>
>
>Dr. David B Hayes
>Boston Biomedical Research Institute
>64 Grove St.
>Watertown, MA  02472
>617-658-7738
>
>Boston Biomedical Research Institute... Today's Research for Tomorrow's 
>Health.
>Please visit us at www.bbri.org
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