Hi Hugh,
You might have accidentally received my last message twice. There was
a hardware failure affecting the Gna! infrastructure
(http://gna.org/forum/forum.php?forum_id=2327). As relax relies on
this infrastructure, the mailing lists were down for about 6 days (as
well as the website, subversion repository, bug tracker, etc.)! If
you sent a message in during the down time, you may have seen a series
of messages such as:
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If so, you will have to resend the original mail.
Regards,
Edward
On 27 February 2012 21:50, Edward d'Auvergne <[email protected]> wrote:
> Hi Hugh,
>
> I'll answer below:
>
>
>> 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.
>
> This is an issue which I don't think anyone has studied in depth and
> comprehensively before. It's also difficult to study as structural
> issues are likely to be a bias (directional randomness) rather than a
> variance (pure randomisation). For example a whole structural element
> could be reoriented. Or there could be domain motions which are not
> taken into account in the current level of theory. There is a lot of
> potential to develop this area of analysis in the future ;)
>
>
>> 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.
>
> HydroNMR, from what I've heard, is terrible at prediction when there
> are domain motions. The program is also not very good at predicting
> the behaviour of proteins at the concentrations you have in the NMR
> tube. It is designed for prediction of the diffusion tensor of an
> isolated molecule, but your molecules are very close together in the
> NMR tube and this has significant consequences.
>
> The lower Dratio is understandable as you have domain motions and the
> core is only partly affected by the other domain. Did you perform an
> analysis with the two domains separately? For example as in my
> analysis at http://www.sciencedirect.com/science/article/pii/S0022283607007073
> for the DsbA oxidoreductase.
>
>
>> 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?
>
> You could treat each domain in a separate model-free analysis. But
> model-free analysis assumes a static, perfectly averaged structure as
> the backbone of the analysis. If you do not have this, i.e. there are
> internal reorientations caused by the domain motions, you then have to
> rely on the local tm models. Though these models can easily absorb
> and hide motions if you have data at only 1 or 2 fields. Or
> alternatively you could consider developing a theory or method of
> analysis to handle this situation.
>
>
>> 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?
>
> If there are Rex terms in the spherical model but not in the
> ellipsoid, then these are almost guaranteed to be false motions (as
> described by Tjandra et al, 1995). The additional ns terms are also
> likely to be fake as described by Schurr 1994. Note that the local_tm
> models could sometimes absorb the Rex values into the local tm value
> as the data for these residues is usually very noisy. The only real
> way to determine if the Rex terms are real would be to perform some
> relaxation dispersion measurements, although that is not always
> conclusive. Data at 3 field strengths is also very powerful for
> determining if the Rex values are real. You must also remember that
> we assume that Rex in a model-free analysis is in the fast exchange
> limit, which is not always the case, and the only way to differentiate
> between quadratic fast exchange and linear slow exchange (and
> everything inbetween) would be to have 3 or more field strength data.
> Or, of course, relaxation dispersion data. I hope this helps.
>
> Regards,
>
> Edward
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