Re: CSA & bond length -- September 29, 2006 - 17:08

Salut Séb, welcome to the relax users mailing list.  Thank you for
responding Alex.  The CSA value is important as the example shows.
However I would call this a 'fringe' example as it represents a highly
restricted nanosecond motion.  The relaxation data for this example
was generated by back calculation using the CSA value of -160 ppm.
Although as Alex pointed out relax is capable of optimising the CSA
value, I would be wary of these models as they are essentially
untested.  I've played around with the models a little and I have a
feeling that the R1, R2, and NOE values are not sufficient to tease
out the CSA.  To test these models using just the R1, R2, and NOE at
multiple field strengths, the CSA would need to be accurately measured
using one of David Fushman's techniques (I'll talk about this next)
and the values compared to those fitted using the models built into
relax.

I believe that the value of -160 ppm was determined by solid state NMR
of small peptides (it's been a few years since I read the litterature
on the CSA value in proteins, so I could be wrong).  However a number
of publications have demonstrated that the average CSA value in
solution is higher.  I would say that the authorative expert in the
field is David Fushman.  The JACS reference you cite is just one of
many of his publications on measuring the CSA.  He has demonstrated,
using I think three different techniques now, that the CSA in proteins
is highly variable.

Idealy for highly accurate model-free analysis, the CSA value should
be determined either prior to or during model-free analysis using one
of his techniques.  However most people appear happy to just set the
CSA value to either the 'ancient' value of -160 ppm or the solution
average of -170 ppm (David's work again).  Using the data you have
currently collected, I would personally use the value of -170 ppm.  Is
the value of -172 ppm from the Hall and Fushman paper you cited?  I
haven't read that paper yet.

Edward


P.S.  I might change the sample scripts to -170 ppm.  I had intended
to change the value a while back but forgot about it.



On 9/30/06, Sebastien Morin <sebastien.morin.1@xxxxxxxxx> wrote:
>  Hi again
>
>  Thanks for your answer !
>
>  I think that, for me, the CSA value would have a significant impact on my
> analysis since my protein has a tumbling time of about 13 ns and I have data
> from 500, 600 and 800 MHz...
>
>  I don't know if this is relevant, but I performed simple tests with the
> test data and sample scripts provided with relax (path :
> 'relax/test_suite/data/model_free/S2_0.970_te_2048_Rex_0.149'
> in version 1.2.7 and the sample script 'mf_multimodel.py')...
>
>  TEST 1
>  =====
>  r = 1.02
>  CSA = -160 ppm
>  m4
>  S2 = 0.97
>  te = 2048
>  Rex = 0.149
>  X2 = 7.3e-28
>
>  TEST 2
>  =====
>  r = 1.02
>  CSA = -172 ppm
>  m4
>  S2 = 0.97
>  te = 82
>  Rex = 4.34
>  X2 = 2.27
>
>  As you can see, for this single residue (with data at 500 and 600 MHz),
> there is no effect for the value of S2, but the effect is important for te
> and Rex... And still, the best model (the lower X2) is m4 for both
> situations...
>
>  I think that this ambiguity in the value for CSA leads to important
> variations in the interpretation of relaxation data.
>
>  Thanks for getting me to understand more this topic and also choose the
> best value to use...
>
>  Séb
>
>
>
>
>  Alexandar Hansen wrote:
> Hi Sebastien,
>
>  I'm quite new to relax as well, but I can give you at least a some answer
> to the questions you pose.
>
>  In general, the CSA mechanism is a little underappreciated.  At low enough
> field strengths for 15N relaxation (400-500MHz), the 15N CSA accounts for
> somewhere between 10-20% of your R1 and R2 rates.  Varying the CSA magnitude
> between 160 and 172 only changes this by 2-3%.  So, if relaxation rates are
> measured with, let's say, 5% error, there's no statistical reason to vary
> the CSA.  As we go to higher fields (800MHz), the CSA can account for 50-60%
> of the R1 and R2 rates and varying the CSA between 160 and 172 can affect
> those rates by up to 10%.  So, now people are finding that this thing called
> CSA is relatively improtant and should be better understood.
>
>  In many analysis techniques, such as relax, you have the option of letting
> the CSA vary.  For relax, I believe that's models m10-m19 and tm10-tm19.
> One word of warning though, I wouldn't encourage fitting the CSA unless you
> have data at multiple field strengths as you're adding another variable to
> the analysis, so the standard 3 measurements at a single field strength are
> likely not enough to do this.  You also run the risk of overinterpretting
> your data because, in my opinion, varying the CSA freely in relaxation
> analysis is not unlike simply throwing in a fudge factor. :-)
>
>  As for what is the best value to use, I can't really help you there. We'll
> have to wait for some of the protein people to respond (I know RNA better
> ;-) ).  But if you're at low enough fields or tiny proteins (<2-3 ns tau( m
> )) it shouldn't really matter what you use.
>
>  I hope all of this makes sense and I haven't said anything blatantly
> incorrect.  If I have, hopefully someone will follow up on both of our
> posts.  Thanks, and good luck!
>
>  Alex Hansen
>
>
>
>
>  Hi
>
> I am new to relax and have a quite general question about the value used
> for the CSA while studying proteins' 15N-1H vectors with model-free
> approach.
>
> In the litterature, we mainly find two values for the CSA (-160 and -172
>
> ppm).
>
> There is, if I understand well, a link between the bond length and the
> CSA, but everyone seems to agree about using the same value of 1.02 A
> which should give rise to a mean S2 of 0.85 for secondary structure when
>
> combined to a CSA of -172 ppm.
>
> In the relax sample scripts (as well as in the Model-free manual), a
> value of -160 ppm is used for CSA.
>
> What is the best value to use and, most importantly, why ?
>
>
> Also, what about the CSA variability from one vector to another (JACS,
> 128 (24), 7855 -7870, 2006) ?
>
> Thanks !
>
>
> Sébastien
>
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>  --
>
>  ______________________________________
>  _______________________________________________
>  | |
>  || Sebastien Morin ||
>  ||| Etudiant au doctorat en biochimie |||
>  |||| Laboratoire de resonance magnetique nucleaire ||||
> ||||| Dr Stephane Gagne |||||
>  |||| CREFSIP (Universite Laval) ||||
>  ||| 1-418-656-2131 poste 4530 |||
>  || sebastien.morin.1@xxxxxxxxx ||
>  |_______________________________________________|
>  ______________________________________
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