[RASMB] sedfit vs SOMO vs structure

[Borries Demeler]

> 
> Hi
> 
> This has prompted me to do a quick check of the idea of 'random' residuals
> and the RMSD of a couple of recently run proteins. I have attached a
> graph of RMSD for a fit (abs 230nm approx 0.7AU, 200 scans fitting every
> scan) for both proteins vs.  successive deletions of the scans. Therefore
> each point is a fit minus the first, second, third etc. scan. As you can
> see the fit is appreciably better after discarding the first few scans
> (nothing new here). The non-random behaviour of the fits in both cases
> however (protein 1 being a much more well behaved protein) does not
> diminish until way after the 23rd scan, with a plateau of RMSD occuring
> somewhere in the 0.00360 region.
> 
> Now, this obviously is not an exhaustive study (just did it in the last
> 10 mins), but interesting non the less. I normaly leave out the first
> few scans, but this may have prompted me to look at leaving out rather
> a few more.
> 
> Cheers
> 
> Tom
> 
> Dr T A Jowitt Biomolecular Analysis Core Facility Manager Faculty of Life
> Sciences, University Of Manchester Michael Smith Building Manchester,
> M13 9PT Tel; +44 (0)161 3065176

Tom,

like you say, "nothing new here". Indeed, I have seen this as well.
When I do, I check to make sure that a) my radial calibration is correct
b) my meniscus position is fitted correctly. These are the most common
reasons for the symptom you describe.

The other possibility of course is improper (or lack) of modeling the
rotor acceleration, or non-ideality in diffusion or refractive problems
with steep gradients not accounted for by your model. Especially for early
scans, the concentration gradient is very steep for large, slow diffusion
proteins, when run at high speed. This may exceed the refractive index
difference the optics can handle in a linear fashion, and give you
artifacts that disappear once the boundary has broadened a bit.

Other than that, the 0.0036 RMSD in absorbance optics is exactly what we
see - 0.0025 when using intensity mode, which buys you a factor of 1.4.
This of course assumes fitting of time- and radially-invariant noise.

Best wishes, -Borries