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
[EMAIL PROTECTED]
