On Dec 10, 2008, at 17:02, Mischa Machius wrote:
On Dec 10, 2008, at 9:52 AM, Mark J. van Raaij wrote:
as a small variation on this, I would first "finish" the protein,
and then include ligands, working from larger to smaller (ATP =>
citrate => glycerol => sulphates => waters). Sometimes several
waters (from automated solvent building) in place of a bona fide
ligand (or a glycerol for example) refine eerily well and give
reasonable maps...
Automated solvent building that includes automatic refinement should
probably be banned ;)
Yep - thats a nice idea. Maybe also ban real space refinement in Coot,
then graphics, and then go back to wire models, with hand-contoured
maps. ;-)
Sorry for that Mischa, just joking, now I will be serious !
I can agree that automated solvent building and refinement can be done
badly - maybe it is being done badly - but that is no reason to
condemn the principle,
or disqualify the procedure as a whole.
Personally, I add ligands before waters in general, I start adding
waters early (yes, with an automated procedure) since most waters are
better visible
than most NZ of Lys residues, and I treat protein, ligands and solvent
as one structure that I am trying to make the best of. I check all the
main chain,
side chain, waters, and ligands of all my structures (and these of my
group ... not many ... but we are working on this) and I often delete
waters
that were placed automatically, to add side chains (double
conformers...), glycerol, sulfates, or whatever makes chemical sense
for the experiment.
Automated solvent building that includes automatic refinement saves me
a great deal of time, although its not flawless and needs
critical interpretation.
Automation is no substitute for a brain, but likewise using your brain
for mostly trivialities is misusing your brain...
Having said these, I will shamelessly admit here (as I think I also
did in the latest Gordon conference?) that I have never found an omit
map to be *really* useful,
and it never told me something I could not see in the 2fo-fc and fo-fc
maps. Have people seen recently (post-likelihood) an omit map
showing something different than the combination of the two 'usual'
maps? If so I would be really interested to see them.
You can send me the mtz and pdb's, and I promise to put the pictures
on a web site together with an apology for writing this paragraph.
A.
I usually add solvent molecules before adding ligands. For one, the
electron density usually improves when adding solvent, so that the
interpretation of the ligands becomes easier. I would recommend to
check every single solvent molecule, i.e., never use automatic
solvent-modeling and refinement (!) routines blindly. Make sure to
remove those solvent molecules that have clearly been placed into
ligand density before doing the actual refinement.
Another potential problem may have to be taken into consideration as
well: depending on the resolution, it can happen that protein side
chains are being moved into ligand density if it is not occupied by
some atoms. In such cases, I use a mixed strategy derived from the
approaches described in my first post.
Best - MM
On 10 Dec 2008, at 16:41, Mischa Machius wrote:
Kathleen - The easiest way is to simply remove the ligand from the
coordinates and refine for a few cycles. Whether that is
particularly meaningful is another question. Better would be to
remove the ligand coordinates, "shake" the remaining coordinates
(i.e., randomly displace them by a small amount), and then refine.
Even better, perhaps, would be to calculate a simulated-annealing
omit map, but AFAIK, you can't use CCP4 for that. IMHO, the best
option is to not include the ligand in the model-building and
refinement processes until all of the protein(s), solvent
molecules, etc. have been properly modeled. I personally tend to
include ligands only at the very end of the modeling/refinement
process, unless there is really no ambiguity. This strategy will
minimize any model bias from the ligand, and it will give you an
omit map by default (until you actually include the ligand). Best
- MM
--------------------------------------------------------------------------------
Mischa Machius, PhD
Associate Professor
Department of Biochemistry
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.; ND10.214A
Dallas, TX 75390-8816; U.S.A.
Tel: +1 214 645 6381
Fax: +1 214 645 6353
On Dec 10, 2008, at 9:30 AM, Kathleen Frey wrote:
Hi Everyone,
Can anyone tell me a relatively easy way to generate an omit
density map for a ligand? I know that CNS can do this, but I was
wondering if there's a CCP4 related program to generate omit maps.
Thanks,
Kathleen
