Hi Monica
You don't need super-high resolution data for sulfur-SAD (only
sufficient to resolve the sulfurs or "super-sulfurs" if you have
cystine bridges) - you just need to collect the data well enough to
have sufficiently big anomalous differences. So although you probably
want to try to get better crystals, it could well be worth your while
collecting the data as well as you can (and not relying on the
"American method"* which is becoming increasingly popular again).
High multiplicity datasets collected so as to reduce the systematic
error would be your friends here - so change things like the crystal
to detector distance (to use different pixels for equivalent
reflections), orientation of the crystal (use a kappa if you have it)...
You may want to read some of the top hits from a google search for
"sulfur sad resolution"
* American method: "We term the new procedure the 'American Method'
for it demands that the shooting be done first and questions asked
subsequently. Tradition has it that this was the normal procedure of
law enforcement in the American Wild West. Some say traditions die
slowly. Certainly it is well suited for dealing with delinquent
crystals in the New World of Synchrotrons.”
J. Appl. Cryst. (1983) 16, 629 – 636, Michael Rossmann & John Erickson
On 5 Oct 2015, at Mon5 Oct 08:52, Monica Mittal wrote:
> Thank you all,
>
> Thanks !! S-SAD is not possible for these crystals, not so good
> diffraction ones. Super high resolution is very difficult to
> achieve. Possibility is around 3 A. I am just thinking of anything
> possible other than soaks.
>
> :)
>
> On Mon, Oct 5, 2015 at 9:50 AM, David Briggs
> <[log in to unmask]> wrote:
> Hi Monica,
>
> How extensively have you attempted to soak with heavy atoms? The
> fact that your crystals are degrading (inferred from OP) in the
> presence of heavy atoms suggests that they are at least interacting
> with the protein. Have you tried short soaks (15mins), lowering the
> concentration of the heavy atom reagent by a factor of 10 (or
> higher), co-crystallisation with the heavy atom?
>
> How many Cys & Met residues do you have? Could you try a sulphur-
> SAD experiment to phase the data?
>
> If you have good enough native data there are programs like
> Acrimboldo ( http://chango.ibmb.csic.es/ARCIMBOLDO/ ) that you can
> try ab-initio phasing with.
>
> HTH,
>
> Dave
>
>
>
>
>
> David Briggs
> about.me/david_briggs
>
>
> On 5 October 2015 at 08:38, Monica Mittal
> <[log in to unmask]> wrote:
> Dear all,
>
> I need a general advice. I don't have a suitable Model (Lets say
> only with a similarity of 15%) and no other model to solve the
> structure by MR. Then i think of anomalous experiments, but soaking
> with heavy metals is not good for the crystals. Since the protein
> is being expressed in Pichia cells, growth in Seleno-media is
> cumbersome. There are no ligands or ions that i can use for
> anomalous scattering of protein crystals. I am trying to express it
> in bacteria but have some issues. What is the option that i can
> choose to solve the structure of such a protein ? Your suggestions
> are highly recommended.
>
> Thanks in advance.
> Monica
>
>
Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick
Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH
Chairman of International Union of Crystallography Commission on
Crystallographic Computing
Chairman of European Crystallographic Association SIG9
(Crystallographic Computing)
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