Andreas,
Thanks for clarifying the situation. I have one other suggestion you can
think about based on your description of a "monomeric hybrid." Since you
are mutating your original yeast protein sequence to human/yeast
chimeras, you could directly model the mutations in Rosetta and skip the
homology modeling. Of course, this assumes you are not introducing
deletions or insertions. This approach will also calculate a DDG for you
automatically.
Your mutation combinations can be defined by creating a "resfile," and a
standard Rosetta command call can automate the entire process.
http://www.rosettacommons.org/tiki/tiki-index.php?page=resfile+example
Based on my own experience, I would recommend the following flags:
-Wpack_only, -soft_rep_design: softens the repulsive term and
accommodates mild clashes between atoms
-ex1, -ex2, -extrachi_cutoff 1: allows rotamers to deviate from their
most probabilistic side chain conformations
-multi_cool_annealer: compared to Rosetta’s standard annealing scheme,
multi-cool simulated annealing lowers the system to a cooler temperature
and considers nearly ten times as many rotamer substitutions before
selecting a low energy structure
-repack_neighbors: mutated residues and adjacent residues are allowed to
relax
Here are some other relevant flag combinations with regard to interface
repacking that you can read up on:
-repack_neighbors and -relax_unbound
-min_interface
Good luck,
Steve
Andreas Förster said the following on 9/12/08 4:42 AM:
Thanks to all who responded to my question regarding the energetics of
a known interface applied to orthogolous dimers.
Steven Darnell asked me for some clarifications. I have the structure
of a homodimer, defined the dimerization interface and substituted the
residues at said interface with those of each of four human orthologs
of the original yeast protein. What I call monomeric hybrids are thus
yeast proteins with different humanized dimerization interface. I
recombine these monomeric hybrids to give four humanized homodimers
and six humanized heterodimers. It is the likelihood that these dimers
form that I'm interested in.
Following Diana Tomchick's suggestion, I had PISA analyze the
interface of the original dimer and learned that it might be worth to
consider one other region besides the main helix.
For modeling of the monomeric hybrid, MODELLER is suggested because it
does simulated annealing by default. To get the energetics, Rosetta
with the "-interface" or "-ddg_only" flags might be a good tool. I
have to read up on the details. An alternative is molecular dynamics
in Gromacs.
Thanks for your help.
Andreas
