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[ccp4bb]: refinement problem



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Dear All,

I'm having a refinement problem and would appreciate any suggestions.   

I have a well-refined, well-behaved 2.2 A MAD data set that the structure was
solved with Se atoms to a crystallographic R of  21.2% and a free R of  25.2%
(269 of a possible 271 amino acids, one phospholipid, 55 waters).  The crystals
are P21, a = 43.914 , b = 73.773 , c = 48.185 , b = 114.732.

My problem is in refining a higher resolution native data set, to 1.9 A.  The
data is fairly isomorphous with the MAD data set, P21, a = 43.718 , b = 73.184
, c = 47.427 , b = 113.842.  All data has been processed with denzo/scalepack
(including the MAD data).  For the high resolution refinement, I took the model
from the MAD data, did a rigid body refinement, simulated annealing, and a
B-individual refinement, manually rebuilt a little, ran a conjugate gradient
minimization -- but my crystallographic R is stuck at about 27%, and the free R
is about 31%.  The 2fomfc maps calculated after this refinement are beautiful.
Truly, without exaggeration.  I can contour to about 2 sig and see holes in my
aromatic residues, and water molecules are clearly visible.  Carbonyl oxygen
bulges are readily apparent throughout the molecule.  I can automatically add
about 100 water molecules, and my crystallographic R drops to 25.0% and a free R
of 28.8%.  

I have tried numerous refinement strategies, including refining with and without
the MAD data set, cutting the resolution to 50-2.2A, cutting the resolution to
10-1.9A, and nothing seems to get me beyond this 25% crystallographic R.  The
maps look, to me at least, way to good to have this high of an R.  The B's
refined with the MAD data and the native data are very comparable.  OMIT maps
showed no obvious problem with the model.

My gut feeling is there is a problem with the data, but it looks good to me.
I'm including the end two tables from the final scalepack run.  The only thing I
can think of with this data, is that it is a synchrotron data set, and during
that data collection run there were considerable problems with the beam (it kept
dumping prematurely). 

I would appreciate any comments or suggestions.


     Shell            I/Sigma in resolution shells:
  Lower Upper      % of reflections with I / Sigma less than
  limit limit     0     1     2     3     5    10    20   >20  total
  50.00  4.09   2.7   4.3   5.1   5.5   6.0   7.9  12.8  85.6   98.4
   4.09  3.25   0.2   0.6   1.1   2.4   4.2   8.6  19.1  80.8   99.9
   3.25  2.84   1.2   2.5   4.8   6.6  11.4  21.2  46.5  53.4  100.0
   2.84  2.58   2.2   4.8   7.8  11.1  18.7  36.8 100.0   0.0  100.0
   2.58  2.39   3.2   7.3  11.9  17.3  26.4  51.5  99.9   0.0   99.9
   2.39  2.25   4.3  10.3  18.1  24.4  35.5  70.0  99.8   0.0   99.8
   2.25  2.14   4.6  11.6  21.8  31.5  48.8  99.6  99.6   0.0   99.6
   2.14  2.05   7.2  16.2  27.5  37.6  56.2  97.9  97.9   0.0   97.9
   2.05  1.97   9.5  19.2  29.5  40.4  58.3  94.5  94.5   0.0   94.5
   1.97  1.90   8.2  20.7  33.3  44.5  66.3  88.4  88.4   0.0   88.4
 All hkl        4.3   9.7  16.0  22.0  33.0  57.4  75.6  22.3   97.9


               Summary of reflections intensities and R-factors by shells
     R linear = SUM ( ABS(I - <I>)) / SUM (I)
     R square = SUM ( (I - <I>) ** 2) / SUM (I ** 2)
     Chi**2   = SUM ( (I - <I>) ** 2) / (Error ** 2 * N / (N-1) ) )
     In all sums single measurements are excluded

 Shell Lower Upper Average      Average    Norm.  Linear Square
 limit    Angstrom       I   error   stat. Chi**2  R-fac  R-fac
      50.00   4.09  2614.5    72.3    36.3  0.981  0.028  0.033
       4.09   3.25  2132.1    61.3    34.0  1.295  0.037  0.040
       3.25   2.84   935.9    42.2    23.6  1.243  0.056  0.055
       2.84   2.58   565.2    40.9    20.0  0.991  0.075  0.071
       2.58   2.39   382.9    35.8    19.4  1.008  0.102  0.093
       2.39   2.25   305.4    36.2    20.9  1.106  0.134  0.121
       2.25   2.14   253.9    41.8    22.6  1.011  0.193  0.000
       2.14   2.05   199.1    38.8    23.7  1.027  0.196  0.170
       2.05   1.97   174.9    37.4    24.4  1.024  0.207  0.177
       1.97   1.90   138.4    37.9    24.0  0.979  0.243  0.221
  All reflections    787.7    44.7    24.9  1.072  0.062  0.044

Thanks,
Marilyn Yoder


Marilyn D. Yoder                                               yoderm@umkc.edu
Division of Cell Biology and Biophysics           phone: 816-235-1986
School of Biological Sciences                          fax:      816-235-1503
University of Missouri-Kansas City
5007 Rockhill Rd.
Kansas City,  MO  64110-2499