There are several scenarios and it is hard to generalise.
Certainties are:
1) The better your data the easier it is to see a ligand. At 3A it can
be hard to model a blob, but it is usually straightforward at 2A. It is
harder if your data is twinned etc..
2) There is a lot of unnecessary and confusing molecular replacement done.
If your ligand data is reasonably isomorphous ( similar cell and space
group) then the berst procedure is to start from your known structure
and do rigid body refinement first to correct for any small changes of cell.
If you do a full blown MR run you are very likely to get a solution on a
different origin - this is formally correct but makes it hard to overlap
the maps!
I often merge the 2 data sets and do a Scaleit check to see how the
differences seem..
3) It is a good idea to inspect the ligand map AND the apo map at the
same time to see what has changed around the active site - it isnt only
ligand you are insterested in - have side chains or waters moved?
4) If desperate, it can help to do an Fobs_lig -Fobs_apo if they are
isomorphous enough -
Eleanor
Schubert, Carsten [PRDUS] wrote:
Aha Dr. Palm ....
Tough to generalize this, but if the ligand is weak or the site only
partially occupied you need to refine almost to completion. Since you
seem to be doing something akin to fragment screening I can only pass
on my experience. I had plenty of cases where the Rfree was ~35 after
a rigid body refinement and the ligand density was practically
indistinguishable from bound water. Refinement to an Rfree of ~25 with
waterpicking/pruning resulted in clear enough density to place the
ligand. This is probably overkill for very tight binders but necessary
for weak binders.
phenix works pretty well for this since one can do rigid body
refinement, individual positional refinement and B-refinement all in
one shot.
Ciao
Carsten
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of
> Palm
> Sent: Tuesday, May 29, 2007 4:25 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] How to determine ligand binding from diffraction
> pattern?
>
>
> I would like to expand on the question and answer below and compare
> your experiences: Looking for ligands in many different soaks
> / cocrystals
> of your protein of interest, you still should do molecular
> replacement
> and a bit of refinement. I agree with Steve, but how much refinement
> is necessary and enough?
>
> We have a specific case with a 24 kDa protein crystallizing in P6522
> with resolution of 2.5 - 3 A, which should be comparable to most
> cases. The ligands have 10 - 20 non-hydrogen atoms (most of the time
> we don't know, we are actually screening for them). How far should
> we refine to see if we have only water molecules or a ligand bound
> - to an Rfree of 0.45 or 0.40 or 0.35?
> greetings
> Gottfried
>
>
> Dear all,
>
> Is there a simple way to determine whether ligand is bound or not
> by comparing the diffraction patterns between ligand-free (structure
> known) and ligand-soaked protein crystals? I would like to solve
> the ligand bound protein structure, but before I do so, I have to
> find out if the ligand is actually bound. Thank you very much!
>
> Best,
>
> Joe
>
> Having done this a few hundred times, I would strongly suggest that
> you just collect the data and solve the structure. Since you already
> have the apo structure solved, then it really isn't that much work
> to do an MR solution on the complex. Be aware that quite frequently
> there is enough non-isomorphism to necessitate partial refinement
> of the "complex" structure before recognizable density will appear
> for the ligand. The definitive answer can only be obtained with
> a full data set, so go for it.
>
> Good luck-
>
> Steve
>
>
>
>
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