Thank you Edward for your prompt response.
This has given me more interpretable numbers. From reading all the
previous correspondance (some of which has touched upon this issue), I
still remain a little concerned as to the dependence of the results on
the input .pdb file. I am almost certainly being overly cautious but
as this is probably a common concern it'd be nice to hear your
thoughts.
The Dratio that has resulted from the fitting of my apo protein is
1.55, as compared to that predicted by HydroNMR (1.75). For HydroNMR
to overestimate values by this much is not itself a surprise or
concern, but it is striking that the predicted Dratio for the closed
complex is closer (1.43). I should emphasize that this is 2-domain
protein with 2 "hinge" regions - one of which is mobile on the ms-us
timescale and thus not amenable to study, and the other has fitted Rex
terms. Infact about a third of the residues studied have fit Rex terms
in the chosen ellipsoid model. On face value this is not actually a
surprise seeing as various parts of the protein are flexible on the
ms-us timescale as evidenced by line-broadening.
It is possible that flexibility of the hinge regions is causing
fluctuations in not only the principle diffusion tensor, but also the
amide bond vectors relative to it. How could one ever test for this?
Obviously the long-winded way is to fit the data with the closed
complex coords and see if the X2 value is lower. But is there
something more sophistocated and quick?
I have looked at residues with fitted Rex terms in the spherical model
which are absent in the ellipsoid model, and also residues with ns Ts
terms in the spherical model which are absent in the ellipsoid. Both
of these have VERY strong dependence on bond vector relative to the
principle diffusion axis, which was unsurprising based on what you
have said elsewhere. Encouragingly this relationship is not so strong
if you compare to the bond vectors taken from the closed structure. On
the other hand, the local_tm model fit very few Rex terms which is a
little concerning. I have looked if the residues with fit Rex terms in
the ellipsoid model but not in the local_tm model have a bond vector
dependence, which they don't. Are there any other consistency tests
you would recommend?
Thanks
Hugh
PS. I should emphasize these crystal structures are both high quality
(Resolution < 1.6 angstroms)
On 3 February 2012 11:41, Edward d'Auvergne <edward@xxxxxxxxxxxxx> wrote:
Hi Hugh,
Welcome to the relax mailing lists! The numbers you quote don't seem
too surprising. The Dr value is very close to zero which means that
the rhombicity is very low. The Da value is hard to judge without
seeing the corresponding tm or Diso value. You could try the
following to obtain a more detailed description of the diffusion
tensor. In relax, assuming you are in the directory of the final
results file, type:
relax> state.load('results.bz2')
relax> diffusion_tensor.display()
For the ellipsoid tensor, you can directly compare the Dx, Dy, and Dz
values as these are the rotational diffusion coefficients in the three
principle directions. For the spheroid tensors, you might find the
Dratio parameter more meaningful. The display for these tensors will
also give you Dpar and Dper which are equivalent to the Dx, Dy, and Dz
eigenvalues and hence can be directly compared. Note that the purpose
of the results file is primarily to store the tensor data at full
machine precision rather than to be read (even though the information
is presented in a readable form). The diffusion_tensor.display user
function is designed to present the tensor in a more understandable
form. I hope this helps.
Regards,
Edward
On 2 February 2012 16:02, Hugh RW Dannatt <h.dannatt@xxxxxxxxxxxxxxx> wrote:
Dear All,
I have just finished fitting some relaxation data with the
full_analysis.py script, which as far I can tell has behaved exactly
as I would have expected, but I'm having trouble working out what the
numbers in the results file mean. My molecule is very anisotropic and
so the diffusion models were (with decreasing AIC) - sphere, oblate,
prolate, ellipsoid. The minimised value of Tm is also what I would
predict from the T1 & T2 values, and also matches the average number
predicted by HydroNMR. Also, the location of Rex terms correlates
nicely with what I would expect from Relaxation Dispersion studies.
Overall I'm very satisfied.
Much as the program has seemed to behave as I would expect, I am still
keen to be able to validate the results by whatever external means
possible. And so I would like to see if the degree of anisotropy fit
agrees with that predicted by HydroNMR (and, less scientifically, what
you would expect by eye!). However, the numbers in the results file
for the ellipsoid fitting seem very odd to me - I get Da = 5321992.00,
and Dr = 0.066925928). The way that they are defined in the manual
would mean that my molecule has one side several thousand times longer
than the other! In the prolate fitting I get a similar Da figure (this
time 5635770.81).
Can somebody explain to me what these numbers mean and how I can
calculate the individual anisotropic diffusion rates? If anybody has
any tips on data validation I'd be keen to hear it also.
Thanks
--
Hugh Dannatt
PhD Student Researcher
Prof. Jon Waltho Lab
Department of Molecular Biology & Biotechnology
University of Sheffield
Firth Court
Western Bank
Sheffield
S10 2TN
0114 222 2729
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--
Hugh Dannatt
PhD Student Researcher
Prof. Jon Waltho Lab
Department of Molecular Biology & Biotechnology
University of Sheffield
Firth Court
Western Bank
Sheffield
S10 2TN
0114 222 2729
Re: Anisotropic Diffusion coefficients