Hello Edward and relax users,
I have been recently using relax to carry out model free analyses on two
different states of my protein at 600 and 800 MHz. I am working with a LOV
domain (132 residues) which exists in a ground "dark" state and a photoactive
"light" state. Both states exist as a symmetric homodimer in solution. For
the analysis I have been using the crystal structures, one monomer at a time.
Also, the resonances showing overlap have not been included in the analyses.
There is no difference in the backbone as such between the two crystal
structures. Along with model-free I have also carried out reduced spectral
density mapping. The J(0)s of both the states agree well between the two
fields. Hence there is nothing wrong with the data as such. Also both the
J(w) agree well with my het-NOE and R1 with respect to regions in the protein
showing fast timescale backbone motion.
The model free analysis of the "dark" state goes fine. The S2 values show
same regions having fast timescale motions as observed from the raw
relaxation data and rSDM. Both the monomers individually give me
approximately comparable results. For e.g., a loop between residues 100 and
110 shows flexibility on fast time scale according to the raw data, rSDM as
well as relax S2 values.
However, for the "light" state the results from model free appear quite
bizarre for both the monomers. The average S2 for the "dark" state was ~ 0.8.
As per my raw data and rSDM, I expected the S2 values to be slightly elevated
in "light" state. However, my "light" state S2 values are on an average 0.17!
Also the same loop is now with higher than average S2 values in the "light"
state whereas the raw data and rSDM showed the loop to be flexible, albeit
less flexible compared to the "dark" state. The internal motions (Te) are
less than 5 ps and seen only in 7 residues. No Ts or Tf. I have attached
figures from the spectral density mapping as well as the "dark" and "light"
state S2 values to illustrate better what I am trying to say.
Also, I repeated the "dark" state model free analysis with "dark" state
structures. thinking that there might be some bug somewhere and got results
just like earlier. But not with the "light" state. Since the backbones
between two states are near identical, I used the "dark" state structure to
run with relax on the "light" state data. I again get these bizarre results.
I am about to run model free with "light" state structure and "dark" state
relaxation data. However, I am not sure where the problem lies, whether it is
the structure, my data or some bug somewhere. Since the J(0)s add up nicely,
I do not think it is the data. It is unlikely that it is bug since the "dark"
state ran fine. It is also unlikely that the structure is strange since dark
and light states have near identical backbones and there are no missing
residues in either of the structures. I must also add that the correlation
time obtained from relax are actually comparable with the ones extracted from
R2/R1 ratio for both the "dark" and the "light" states. It is only the motion
parameters which are weird for the "light" state.
Has anyone encountered something like this recently? Any help/advice here
will be very helpful. At this moment the data is only with two fields but I
do plan to run relaxation at 900 MHz and use the three fields for model-free
as soon as I get time on the magnet.
Regards,
Vineet Panwalkar
Institute of Complex Systems-6: Strukturbiochemie (ICS-6)
Building 05.8v, Room 2022
Forschungszentrum Jülich
52425 Jülich
Germany
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Forschungszentrum Juelich GmbH
52425 Juelich
Sitz der Gesellschaft: Juelich
Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498
Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher
Geschaeftsfuehrung: Prof. Dr.-Ing. Wolfgang Marquardt (Vorsitzender),
Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt,
Prof. Dr. Sebastian M. Schmidt
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Model free on symmetric homodimer, problems with grace plots