Re: [ccp4bb]: refinement with higher resolution data

["Eleanor J. Dodson" <ccp4@ysbl.york.ac.uk>]

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GUAN wrote:
> 
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> dear all:
> This is a refinement problem with higher resolutiuon data.
> Once the 2.4 A structure  was solved, and now I have collected 1.4 A data for
> the same crystal form. The cell constants differ very little. The sequence is
> not known, but some homologous sequences exist. Two molecules in the A.U., but
> I have not found the NCS aixs.



 My advice relates to CCP4 programs but other software can do the same
operations

 You can find the NCS operator by LSQKAB 
fit molecule 1 to molecule 2 and that gives you the rotation matrix and
translation vector needed.

 However if you use REFMAC then this is determined within the program.
You just need to specify which domain matches which.

> I use the 2.4A structure (water deleted, B factor reset 15.00) as initial model
> to do MR with the new 1.4 A data. The data is very good (Rmerge is 4.0% for the
> whole data set and 20% for last shell. I am using CNS to refine it.(just did rigid.inp
> and anneal.inp, and the R and Rf were only lowered a little, R~35%, Rf~36%).


 Before doing much map searching I would certainly refine what you have.
 Improving the B values residue by residue will lower the R factor and
improve the map quality.

with 1.4A  data you hardly need an omit map; the ML weighted map should
be quite OK for correcting any side chains and if you contour at a LOW
LEVEL, should show up the missing residues if they are at all ordered.
Remember missing residues will be ~ 1/2 height of those you phased on..

 You may improve the map by averaging the density for the two molecules;
you can do this with MAPROT using the rotation matrix etc from LSQKAB.


 To verify poor looking residues, you can set all their occupancies to
0.00 and see what the maps look like without them. That is a sort of
controlled omit map I guess..

 I dont think it is sensible to place waters near the missing stuff till
you have tried to fit the peptide.

 Adding other clear waters in remote parts of the map can help improve
the phasing though.

 You could try Arp-Warp - this is sort of automated building, and would
prob work at your resolution..
Of course it too will find the well defined parts first..