Hi Jeremiah,
On Sat, Jan 09, 2010 at 05:01:07AM +0000, Jeremiah Farelli wrote:
> Does anyone know of an example where SHARP was used to phase
> multiple sulphur-SAD datasets and combine all of the data into one
> map?
I'm not aware of exactly such an example - but there shouldn't be any
problem with that at all. SHARP can easily handle that. Since it
doesn't need that distinction of 'native' and 'derivative' it would
just be a case of several compounds with different S-sites.
> I have multiple crystals of the same protein, but each crystal form
> has a different LtoM mutation (the sulfur content of the native
> construct is too low for phasing without the mutations). The
> crystals are sensitive to oxidaton, so I cannot make one construct
> with all of the mutations, and selenomethionine incorporation is out
> due to the oxidaton issues. Each crystal form from all mutants is
> isomorphous.
>
> I would like to collect highly redundant datasets of each form, and
> then use SHARP to find all the Sulphurs, and combine the initial
> phases from all datasets with the native data and build the model.
Remember that SHARP doesn't find the sites - autoSHARP could try
finding them by using SHELXC and SHELXD (from G. Sheldrick).
> The protein has two domains, and I know the structure of one of them
> (~40% of the total), so I should be able to use the intial MR phases
> to help with finding the sulphurs....
That might be a good starting point: it is very easy to include the MR
phases into a SHARP run ('external phase information') and if those MR
phases are half decent your S-sites should be visible in the
log-likelihood gradient maps.
This is a feature available in SHARP for a very long time - see
http://www.globalphasing.com/sharp/manual/chapter2.html#external
> Is this feasible?
Depends on your signal, non-isomorphism, radiation damage etc.
> Are there any examples like this out there?
Well, we've used the MR-phases plus experimental phasing for more than
a decade now - seems such an obvious idea that we never thought of
writing a 'proper' paper about it.
Cheers
Clemens
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