[RASMB] recommended max rmsd for achieved equilibrium

[Arthur Rowe]

Hi Frank

I wonder why you are running so unusually long a column? I calculate it to be 6.2 mm in length, which will inevitably mean the distribution takes a very long time indeed to attain equilibrium. Using a little Excel utility I wrote a while back, which among other things computes using the old formulae the "time to equilibrium within 0.1%", if your trimer has a MW of 200,000 then it will take at least 147 hours to get there! Although if it is a trimer of a 30 kDa monomer, then a mere 108 hours might suffice. That assumes your trimer to be more or less globular - if the frictional ratio is > ~1.2, then the time to equilibrium increases. Of course with half the column length - and a 3.1 mm column gives you lots of data points - then the time to equilibrium goes down 4-fold! And a 3.1 mm column is quite long by most standards, and gives you lots of data points (OK - that applies to fringe optics, less to absorption).

Doing matching of successive scans, then you would hope to see the rmsd between successive to be of the same order as what we find when we know the two scans to be identical, which is around 0.007 fringe*. I mean, if two successive scans differ by no more (judged by rmsd) than would two identical scans, you are home and dry.

Best of luck!

Arthur

*for a discussion of the nature and magnitude of noise in interference/absorption signals see Ang & Rowe (2010) "Evaluation of the Information Content of Sedimentation Equilibrium Data in Self-Interacting Systems". Macromolecular Bioscience 10 798-807

On 11 Oct 2013, at 15:23, Frank Niesen wrote:

> Hello everybody,
> 
> I was hoping for advice on what rmsd between successive scans at one speed I should be aiming for to determine if equilibrium is achieved? Below 0.01? Below 0.005? Or dependent on how the residuals are distributed?
> To give an example, I am spinning samples of a protein that forms trimers in 2-sector cells (190 ul/cell). The highest concentrated sample is 200 uM and I waited for about 90 hours, seeing gradual improvement, to currently 0.019 rmsd (I use Sedfit to match, thanks Peter & Co. for the excellent tool!). The differences toward the meniscus and the bottom are about -0.03 and 0.04, respectively. So, the distribution is still "developing"...
> But, how good is good enough, having in mind the demand for answers by my collaborators and my desire to proceed to the next speed for the weekend? ;o)
> 
> Many thanks for any help you can give, and a great weekend for all!
> 
> Frank
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Arthur J Rowe
125 Uplands Road
Oadby
Leicester
LE2 4NW

Tel: +44 116 2714502
arthur.rowe at connectfree.co.uk


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