[RASMB] SE things
Holger Strauss
Holger.Strauss at nanolytics.de
Mon Jun 2 00:31:17 PDT 2008
Hi Eric, hi all,
just a few additional comments to the nice discussion going on while others
were asleep.
- what are the Kds for both your SE-models? Is there enough (i.e. > 5%) of
each oligomer in solution? How do the partial concentrations of each
oligomer compare over your examined concentration range? Ultrascan has a
nice feature to show this. This can help to visualize what the statistics
mean in physical terms and if you stand a realistic chance of describing an
oligomer at all. Also, how certain are you of your sigma-value (i.e. vbar
and density)? If you fix the molar mass to the expected value, it might be
off by a couple of percent and this discrepancy will go into the other
exponential terms (Kd and stoichiometry). Can you fit it along with your
Kds for the different models? Or vary it over a +/- 5% range and see what
happens?
- c(s) at "high" concentrations (2-5 mg/mL, depending on the protein) gives
artefactual peaks at higher and lower s-values. They have no physical
meaning but the boundary becomes distorted in ways a single f/f0 can't
describe; it will tend to increase, though, which is a good indication that
something "more" is happening. Likewise, f/f0 < 1.2 (even <1) is an
indication of self-association. (Before everyone rushes to makes statements
about how difficult it is to interpret f/f0 and solvation and hydration and
shape and reaction boundaries and what not: There's information in this
value and it can be used to build hypotheses - with care and not as a
single parameter).
- If you need to show that your crystal-clear trimer is also present in
solution, you might want to consider plotting the local molar masses (from
lnc/r2 of the SE-gradients) vs. concentration and see if the values at the
highest concentrations increase above the expected value of the dimer. If
they do, you can at least rule out the 1-2-only model; could still be a
1-2-4/1-2-3/... Overlaying those plots from different experiments will also
tell you wether your system is fully reversible or not and hence test the
applicability of the Law of Mass Action.
- It could also help to put more information into single SE-gradients, i.e.
increasing the column lenght and scanning with interference optics (still
using multiple speeds & concentrations). You can cover 3 orders of
magnitude on the concentration scale in a single gradient this way and the
fitter will like this. It's also good for the lnc/r2-plots. And it's very
good to keep the centrifuge spinning for days and days.
I have found with other samples that if I increase my protein concentration
much more than 1 mM, my molecular mass will continue shifting towards the
aggregate. With the 1 mM data I have now, the trimer is the main species,
but this shifting trend appears to be starting in this sample, and so I
fear that increasing my concentration may not help clear anything up.
You can use dc/dt- or ls-g*(s) and plot the average s-values. They are more
robust against artifacts than the c(s)-approach and compare very well with
those from c(s).
Using my crystal structure, how can I calculate a theoretical s value for
my trimer? If I use the calculator in sedfit, it generally overestimates
the s value of the monomeric protein which I have observed experimentally.
This isn't surprising since the protein is rod-shaped and not spherical,
but it makes interpreting the predicted s values from sedfit rather difficult.
Hydropro by Garcia de la Torre is the program everybody is using to get
hydrodynamic values from the crystal structure, but there others around
(Solpro, Beads).
Cheers, Holger
=================================
Nanolytics
Gesellschaft fuer Kolloidanalytik mbH
Dr. Holger Strauss
Am Muehlenberg 11
D-14476 Potsdam
Tel.: +49 331 581 8360
Fax: +49 331 581 8361
e-mail: Holger.Strauss at nanolytics.de
Internet: www.nanolytics.de
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