[ccp4bb]: MAD phasing problem (many sites, radiation decay)

[Bjoern Mamat <Bjoern.Mamat@mpibp-frankfurt.mpg.de>]

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We have the following situation:

We did a 3 wavelength Se MAD experiment with a 40 kDa protein (12 Se per
molecule) which is a trimer in solution and either a trimer or dimer in
the asu (space group P21). The crystals are rather weakly diffracting
(usually they do have a sharp resolution limit around 2.8-3.0 A) so we
went to a very strong beam line (ID29, ESRF).

The problem with data collection arised that the crystal showed strong
decay in the beam so that we obtained a good peak, a weaker edge and a
very poor remote data set.

Automatic  Patterson methods failed in yielding the Se sites, but when
using the peak data we could gain a (partial) substructure in SnB and
SHELXD -- 31 sites were the same in both programs. We tried to put
different numbers of sites in the refinement with SHARP (and MLPHARE as
well), and generally the "numbers" looked quite well.  There is a
consensus set of about 25 siteswhich perform rather well in refinement
and can be relocated by difference fourier.

But still the density maps are very poor and don't fit nicely to the
sites -- interestingly, the density looks more than a trimer, the sites
seem to impose two-fold symmetry (if any).

I wonder if anyone has experience with a comparable problem (large
number of size, moderate resolution, low-redundant data, radiation
decay) and can give  me some hints how to get most out of the data or
what kind of additional data collection might be most promising.
(e. g., collecting 3 wavelengths at a weaker beamline, collecting native
data for SIRAS etc.)

Could SeMet oxidation cause a problem like this (poor phases although
the number of correct sites is rather high)?

Thanks in advance for any comments and suggestions

-- bjoern



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Bjoern Mamat                                    mamat@mpibp-frankfurt.mpg.de
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Biotechnologische Studenteninitiative e. V.     http://www.bts-ev.de
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