I used the "comparative modeling" algorithm of the online tool ROBETTA to model a protein structure (http://robetta.bakerlab.org/). The tool always generates five models, to which the ROBETTA FAQs state "In the case of homology modeling predictions, the models are ordered by the likelihood of the alignment clusters. Each alignment cluster represents a particular topology."
Thus, if I understand it correctly, the model 1 will be more likely to be the "true structure" than model 2 and model 2 will be more likely to be the "true structure" than model 3 and so on. I am wondering whether this is quantifiable or with other words: I want to know HOW MUCH MORE LIKELY is model 2 than model 1. Is it possible to get "some numbers" for this?
I have a second problem. I generated a mutant of the protein that I am trying to model and the mutation (one single point mutant) is a complete loss-of-function mutant in vivo. When I run the comparative modeling algorithm on ROBETTA for the wildtype protein I get a structure that looks very plausible (for a lot of reasons) and it is the model ranking second ("model 2"). If I use the "mutant" with the "loss-of-function" mutation" under the same conditions in the same algorithm, I also get this structure (or something VERY SIMILAR). However, this structure is now not model 2 but instead model 4. It seems to be "ranking lower" (i.e. be "less likely"?). Can I interpret these data in a way that the loss-of-function mutation makes it harder to acquire this specific fold (e.g. can I at least say the data hint in this direction)? And if yes, would it be possible to quantify this (i.e. saying HOW MUCH does the mutation interfere with folding)?
Dear Ralf,
The first question that needs to be asked is whether you have simulated your protein in an appropriate force field following modelling apart from the inbuilt ROBETTA algorithms.
Further the ranking of the structures may not be directly related to the induction of a mutation. Try inducing the point mutation in the structure itself using SWISS PDB viewer /DEEPVIEW.
Further analyse the RAMACHANDRAN plot and QMEANS server for model evaluation.
Hope this helps
Hi Ganguli,
thanks for your kind response. Regarding the molecular dynamics simulation, I have not done that yet. I am not a structural biologist, so I am not sure whether I would have the expertise for such analysis. Do you know of any fairly straightforward online tool that is intuitive to use for non-experts, where I could try this?
Regarding the mutation, I have not tried SWISS PDB viewer, but I downloaded Modeller and a friend showed me how to take the "promising" model of the wildtype protein, then mutate one amino acid in the respective structure, and then energy-minimize. I actually have many individual (in vivo) loss-of-function mutations (not just the one mentioned in my original question), but non of these mutations caused the structure to collapse (or change significantly) in this modeling approach. I am worried a bit, however, that this procedure pre-assumes that the "mutant protein" is able to fold into the exact same structure as the "wildtype protein", to then collapse afterwards. But is it not possible that the "mutant protein" would go down a different folding pathway right from the start und would never end up in a "wildtype-like" fold?
You mentioned the Ramachandran plot for my model. It is not perfect (I think three amino acids were outside the preferred region). I showed the plot to a structural biologist, and he said it "looks ok", though (, whatever that means). I haven't tried QMEANS yet. Thanks for the suggestion.
Hi Ralf,
If the size of your protein is less than 400 amino acids you can give a try on CABS Flex server.
3 amino acids is not much of a deviation so I concur with the structural biologist.
Regarding the folding cascade of the mutant protein it is difficult to comment offhand but I believe if the mutation is in a structurally important region for the protein, then it might assume a completely different folding pathway in comparison to its wild type.
Thanks Ganguli,
this is really helpful! My protein is smaller than 400 aa. I will try the CABS-Flex server.
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