two protein complex - or not?

Linear (OLIGOMER), and non-linear (MIXTURE) analysis, singular value decomposition (SVDPLOT), addition of missing fragments (BUNCH, CORAL), analysis of flexible systems (EOM/RANCH & GAJOE), flexible refinement of high-resolution models (SREFLEX)
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Jak
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two protein complex - or not?

#1 Post by Jak » 2010.10.11 15:11

Hi,
I have SAXS data of a two protein mixture. I would like to find whether a complex was formed or if there is no interaction.
The P(r) suggests a complex is formed, as does Rg.
Dammin shape looks like a feasible complex.

I also have SAXS data for each of the two proteins individually. Individual shapes can be fitted into the complex envelope. But does this prove that there is a complex formed or would you get that result anyway if there is no interaction?

What is the best analysis approach to prove complex formation or not, and shape analysis when there are 2 different polypeptide chains?

Thanks
Jak

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Hayds
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#2 Post by Hayds » 2010.10.14 23:00

Dear Jak,

Did you run a concentration series on your "complex" sample (and maybe various molar ratios)? You should be able to follow changes in Rg, Dmax and volume. This should help you to determine if you form the complex or not. You also indicate that you have data for the individual components so presumably you have Rg, Dmax and volume for these also?

I would only trust shape analysis for this situation once you prove that a complex is formed above a certain concentration or particular molar ratio of components.

There has been some pioneering work done on extracting ab initio structures from SAXS data of mixtures, see:

Blobel J, Bernadó P, Svergun DI, Tauler R, Pons M. Low-resolution structures of transient protein-protein complexes using small-angle X-ray scattering, J Am Chem Soc. 2009 Apr 1;131(12):4378-86.

You could also try and run OLIGOMER. If you have structures of the components you can use either calculated curves from the PDB files or the SAXS curves as the input form-factor files (in the case of the experimental curves you may need to do some normalising though, I point you to the OLIGOMER help page for this). You will need to create a model (or a few) for the complex and also calculate a scattering curve for use as the form-factor of the complex. Then you can try and characterise an association equilibrium using the SAXS data collected for the complex at multiple concentrations. This can be a bit tricky and you need to back any conclusions made up with any other data you may have.

Hope it helps,

Haydyn

Jak
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Posts: 25
Joined: 2007.10.01 13:01
Location: UK

#3 Post by Jak » 2010.10.14 23:59

Thanks, Haydyn, for a comprehensive reply.

We did run a concentration series of the complex, but only at a single molar ratio. A rather obvious omission with hindsight, but necessary due to time constraints.
Thanks, too, for the nudge towards OLIGOMER - I wasn't sure from the manual that it was appropriate.

Jackie

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