Andreas,
Here's my $0.02. Would you mind clarifying a few things for me?
> I am working on (the theoretical side of) a protein complex whose
> structure has been solved. The protein homo-dimerizes, mediated
> primarily by two long helices.
So you have a structure of a homodimer...
> Using sequencing alignment and the WHAT IF server, I built monomeric
> hybrid models containing the bulk of the known structure and the
> dimerization helices of homologous proteins. Naturally, I want to
> know how likely they are to form dimers.
Could you explain what you mean by "monomeric hybrid"? I'm guessing you
want to thread two copies of monomer B onto the backbones of homodimer A.
> To look at the energetics, I've run the phenix geometry regularization
> algorithm to minimize clashes and side chain energies.
I've never used phenix and I don't know what sort of search function it
uses. If it's a deterministic algorithm, like dead end elimination,
you'll get the global minimum energy conformation with one run (if it
converges, that is). If it's a stochastic algorithm, like Monte Carlo,
you'll never know if you're at the global minimum. Your best bet is to
run multiple independent minimizations, say 50-100 for starters, and
pick the conformation with the lowest energy score. I'm betting its the
latter.
> The backbone conformation only changes minimally. Next I calculated
> in Rosetta the energetic scores of the models before and after
> regularization and compared with that of the native structure. This
> gave me some numbers that are not inconsistent with experiments.
The following assumes I correctly stated your design problem. Rosetta
does not account for conformational entropy, so the closer the backbones
are between the homodimer A and modeled homodimer B to one another, the
better. You might want to consider fixing the backbones during
minimization.
Also, I don't understand the purpose of calculating the energy of the
non-optimized structure. I would be more interested in the change in
binding energy between the bound and unbound state of the minimized
structure. Rosetta can calculate that in "-interface" mode. There's a
flag to keep Rosetta from performing any design calculations; I think
its "-ddg_only" or something like that. Note that this calculation
assumes the monomers behave like rigid bodies.
Finally, I would minimize homodimer A the same way you minimize modeled
homodimer B as a control, then use Rosetta to calculate its change in
binding energy. Side chain flips of His, Asn, and Gln will make a big
difference. This will give you a number to compare to your modeled dimer.
> Before I sit down and write this up, I wanted to ask the community if
> what I've done makes sense and if there are alternative methods for
> minimizing and calculating interface energies. I don't necessarily
> need docking algorithms as the interface is known. I just want to get
> an energetic description.
If it were me, I would create the homology model using MODELLER (it uses
simulated annealing by default), minimize/relax the structure using
Rosetta, then calculate the change in binding energy with Rosetta.
Remember to repeat stochastic processes. The 50-100 time guideline was
given to me by Deanne Sammond, as in:
Sammond DW, Eletr ZM, Purbeck C, Kimple RJ, Siderovski DP, Kuhlman B.
Structure-based protocol for identifying mutations that enhance
protein-protein
binding affinities.
J Mol Biol. 2007 Aug 31;371(5):1392-404. Epub 2007 Jun 8.
PMID: 17603074 [PubMed - indexed for MEDLINE]
> Thank you.
No worries. Does any one else have any suggestions or corrections?
I've only had 7 hours of sleep since Saturday.
~Steve
--
Steven Darnell
Univeristy of Wisconsin-Madison
Madison, WI USA
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