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

[Quan Hao <qh22@cornell.edu>]

***  For details on how to be removed from this list visit the  ***
***          CCP4 home page http://www.ccp4.ac.uk         ***

Hi, Bjoern,

How does the self-rotation function look like? It should give you a good
indication whether it is a trimer or dimer. 

I would use the peak data only and ignore the other poor data (however,
this is just my personal opinion ...).

Quan 

Bjoern Mamat wrote:
> 
> ***  For details on how to be removed from this list visit the  ***
> ***          CCP4 home page http://www.ccp4.ac.uk         ***
> 
> 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
> 
> --
> ----------------------------------------------------------------------------
> Bjoern Mamat                                    mamat@mpibp-frankfurt.mpg.de
> ----------------------------------------------------------------------------
> Max-Planck-Institute of Biophysics              http://www.biophys.mpg.de
> Heinrich-Hoffmann-Str. 7                        phone  +49 69 96769 384/420
> 60528 Frankfurt/Main, Germany                   fax    +49 69 96769 423
> ----------------------------------------------------------------------------
> AG Praktika                                     AGPraktika@bts-ev.de
> Biotechnologische Studenteninitiative e. V.     http://www.bts-ev.de
> ----------------------------------------------------------------------------

-- 
-----------------------------------------------------------
Quan Hao, PhD                       Tel: 607-254-8983
Associate Director, MacCHESS
Wilson Synchrotron Laboratory       Fax: 607-255-9001
Cornell University, NY 14853-8001   Email: qh22@cornell.edu
-----------------------------------------------------------