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  *  21        tP          7.3      53.5  53.5  41.2  90.1  90.1  90.3    0  1  0  0  0  0 -1  0 -1  0  0  0
  *  21        tP          7.3      53.5  53.5  41.2  90.1  90.1  90.3    0  1  0  0  0  0 -1  0 -1  0  0  0
     39        mC        249.8    114.5  41.2  53.5  90.1  90.3  69.0    1 -2  0  0  1  0  0  0  0  0  1  0
     39        mC        249.8    114.5  41.2  53.5  90.1  90.3  69.0    1 -2  0  0  1  0  0  0  0  0  1  0
and further down lists
indicating at most tetragonal symmetry, shortly after this calculates R-factors for these lattices:
  SPACE-GROUP        UNIT CELL CONSTANTS            UNIQUE  Rmeas  COMPARED  LATTICE-
  SPACE-GROUP        UNIT CELL CONSTANTS            UNIQUE  Rmeas  COMPARED  LATTICE-
   NUMBER      a      b      c  alpha beta gamma                            CHARACTER
   NUMBER      a      b      c  alpha beta gamma                            CHARACTER
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After his comes the table that tells us the quality of our data:
After his comes the table that tells us the quality of our data:
       NOTE:      Friedel pairs are treated as different reflections.
       NOTE:      Friedel pairs are treated as different reflections.
   
   
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     2.04        5134    1601      2347      68.2%    274.7%    291.2%    4913    0.40  325.5%  400.7%    1%  0.608    606
     2.04        5134    1601      2347      68.2%    274.7%    291.2%    4913    0.40  325.5%  400.7%    1%  0.608    606
     total      91819  13782    14656      94.0%      5.7%      5.9%    91589  20.24    6.2%    15.0%    12%  0.897    6450
     total      91819  13782    14656      94.0%      5.7%      5.9%    91589  20.24    6.2%    15.0%    12%  0.897    6450
NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  93217
NUMBER OF REJECTED MISFITS                            1391
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS                  0
NUMBER OF ACCEPTED OBSERVATIONS                      91826
NUMBER OF UNIQUE ACCEPTED REFLECTIONS                13784
So the anomalous signal goes to about 3.3 A (which is where 30% would be, in the "Anomal Corr" column), and the useful resolution goes to 2.16 A, I'd say (pls note that this table treats Friedels separately; merging them increases I/sigma by another factor of 1.41).
So the anomalous signal goes to about 3.3 A (which is where 30% would be, in the "Anomal Corr" column), and the useful resolution goes to 2.16 A, I'd say (pls note that this table treats Friedels separately; merging them increases I/sigma by another factor of 1.41).
For the sake of comparability, from now on we use the same axes (53.03 53.03 40.97) as the deposited PDB id 2QVO.


We could now modify XDS.INP to have
We could now modify XDS.INP to have
  JOB=CORRECT  ! not XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
  JOB=CORRECT  ! not XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
  SPACE_GROUP_NUMBER=  77
  SPACE_GROUP_NUMBER=  77
  UNIT_CELL_CONSTANTS=    53.10    53.10    40.90 90.000  90.000  90.000
  UNIT_CELL_CONSTANTS=    53.03  53.03  40.97 90.000  90.000  90.000
and run xds again, to obtain the final CORRECT.LP and XDS_ASCII.HKL with the correct spacegroup, but the statistics in 75 and 77 are the same, for all practical purposes (the 8 reflections known to be extinct do not make much difference).
and run xds again, to obtain the final CORRECT.LP and XDS_ASCII.HKL with the correct spacegroup, but the statistics in 75 and 77 are the same, for all practical purposes (the 8 reflections known to be extinct do not make much difference).


Following this, we create XDSCONV.INP with the lines
Following this, we create XDSCONV.INP with the lines
  SPACE_GROUP_NUMBER=  77  ! can leave out if CORRECT already ran in #77
  SPACE_GROUP_NUMBER=  77  ! can leave out if CORRECT already ran in #77
  UNIT_CELL_CONSTANTS=  53.10 53.10 40.90 90 90 90 ! same here
  UNIT_CELL_CONSTANTS=  53.03  53.03  40.97 90 90 90 ! same here
  INPUT_FILE=XDS_ASCII.HKL
  INPUT_FILE=XDS_ASCII.HKL
  OUTPUT_FILE=temp.hkl CCP4
  OUTPUT_FILE=temp.hkl CCP4
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===dataset 2===
===dataset 2===
This works exactly the same way as dataset 1.
This works exactly the same way as dataset 1. The table in CORRECT.LP is
 
      NOTE:      Friedel pairs are treated as different reflections.
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION    NUMBER OF REFLECTIONS    COMPLETENESS R-FACTOR  R-FACTOR COMPARED I/SIGMA  R-meas  Rmrgd-F  Anomal  SigAno  Nano
  LIMIT    OBSERVED  UNIQUE  POSSIBLE    OF DATA  observed  expected                                      Corr
    6.06        3925    547      560      97.7%      3.0%      3.3%    3922  56.13    3.3%    1.4%    80%  1.874    242
    4.31        7498    1000      1000      100.0%      2.8%      3.4%    7498  56.91    3.0%    1.2%    65%  1.473    469
    3.53        9407    1291      1291      100.0%      3.4%      3.5%    9407  52.39    3.7%    1.6%    55%  1.276    616
    3.06      11005    1526      1526      100.0%      4.1%      3.9%    11005  42.13    4.4%    2.2%    39%  1.211    732
    2.74      12569    1701      1701      100.0%      5.7%      5.7%    12569  28.38    6.1%    3.7%    4%  0.881    822
    2.50      14020    1904      1904      100.0%      9.0%      9.9%    14020  17.92    9.7%    6.3%    3%  0.741    921
    2.31      15101    2081      2081      100.0%      17.0%    19.0%    15101    9.83    18.3%    12.7%    -5%  0.682    1011
    2.16      11693    2080      2202      94.5%      39.4%    40.8%    11682    4.00    43.6%    45.8%    10%  0.791    1003
    2.04        5152    1607      2345      68.5%      85.6%    93.5%    4943    1.21  101.3%  129.6%    10%  0.718    615
    total      90370  13737    14610      94.0%      4.2%      4.5%    90147  24.22    4.6%    7.3%    22%  0.956    6431
NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  92690
NUMBER OF REJECTED MISFITS                            2318
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS                  0
NUMBER OF ACCEPTED OBSERVATIONS                      90372
NUMBER OF UNIQUE ACCEPTED REFLECTIONS                13738
 
Dataset 2 is definitively better than dataset 1.


==SHELXC/D/E structure solution==
==SHELXC/D/E structure solution==


This is done in a subdirectory of the XDS data reduction directory (either dataset "1" or "2", and we can also try it in a xscale subdirectory). Here, we generate XDSCONV.INP (I used MERGE=TRUE, sometimes the results are better that way) and run xdsconv and [[ccp4com:SHELX_C/D/E|SHELXC]]:
This is done in a subdirectory of the XDS data reduction directory (either dataset "1" or "2", and we can also try it in a xscale subdirectory). Here, we generate XDSCONV.INP (I used MERGE=TRUE, sometimes the results are better that way) and run xdsconv and [[ccp4com:SHELX_C/D/E|SHELXC]].
<pre>
<pre>
#!/bin/csh -f
#!/bin/csh -f
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shelxc j <<end
shelxc j <<end
SAD  temp.hkl
SAD  temp.hkl
CELL 53.10 53.10 40.90 90 90 90
CELL 53.03 53.03 40.97 90 90 90
SPAG P42
SPAG P42
MAXM 2
MAXM 2
end
end
</pre>
This writes j.hkl, j_fa.hkl and j_fa.ins. However, we overwrite j_fa.ins now (these lines are just the ones that [[ccp4com:hkl2map|hkl2map]] would write):
This writes j.hkl, j_fa.hkl and j_fa.ins. However, we overwrite j_fa.ins now (these lines are just the ones that [[ccp4com:hkl2map|hkl2map]] would write):
<pre>
<pre>
cat > j_fa.ins <<end
cat > j_fa.ins <<end
TITL j_fa.ins SAD in P42
TITL j_fa.ins SAD in P42
CELL  0.98000   53.10   53.10  40.90   90.00  90.00  90.00
CELL  0.98000 53.03   53.03  40.97   90.00  90.00  90.00
LATT  -1
LATT  -1
SYMM -Y, X, 1/2+Z
SYMM -Y, X, 1/2+Z
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  shelxd j_fa
  shelxd j_fa


This gives best CC All/Weak of 36.74 / 21.55 for dataset 1, and best CC All/Weak of 35.61 / 26.03 for dataset 2, and .  
This gives best CC All/Weak of 37.28 / 21.38 for dataset 1, and best CC All/Weak of 37.89 / 23.80 for dataset 2, and .  


Next we run G. Sheldrick's beta-Version of [[ccp4com:SHELX_C/D/E|SHELXE]] Version 2009/4:
Next we run G. Sheldrick's beta-Version of [[ccp4com:SHELX_C/D/E|SHELXE]] Version 2011/1:


  shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b  
  shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b  
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  shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b -i
  shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b -i


One of these solves the structure, the other gives bad statistics.
One of these (and it's impossible to predict which one!) solves the structure, the other gives bad statistics.


Some important lines in the output: for dataset 1, I get
Some important lines in the output: for dataset 1, I get
  78 residues left after pruning, divided into chains as follows:
A:  78
CC for partial structure against native data =  36.54 %
...
Estimated mean FOM and mapCC as a function of resolution
d    inf - 4.49 - 3.55 - 3.10 - 2.81 - 2.61 - 2.45 - 2.32 - 2.22 - 2.13 - 2.03
<FOM>  0.763  0.784  0.743  0.682  0.632  0.620  0.621  0.600  0.519  0.416
<mapCC> 0.890  0.936  0.916  0.893  0.838  0.827  0.847  0.858  0.836  0.768
N        721    728    722    720    719    738    749    721    674    721
Estimated mean FOM = 0.639  Pseudo-free CC = 65.26 %
Density (in map sigma units) at input heavy atom sites
  Site    x        y        z    occ*Z    density
    1  0.0293  0.3394  0.3145  16.0000    19.09
    2  -0.1598  0.3789  0.3723  12.7456    15.78
    3  -0.1413  0.4707  0.3704  9.4720    7.85
    4  -0.2238  0.1590  0.4520  9.2176    9.96
    5  0.0387  0.4228  0.3134  1.6608    1.28
Site    x      y      z  h(sig) near old  near new
  1  0.0293  0.3392  0.3148  19.1  1/0.02  2/10.34 4/11.66 4/11.66 5/12.88
  2 -0.1564  0.3740  0.3757  16.4  2/0.35  5/4.38 4/5.45 1/10.34 3/12.03
  3 -0.2146  0.1625  0.4495  11.0  4/0.53  2/12.03 5/15.84 1/16.92 4/17.39
  4 -0.1386  0.4748  0.3671  8.1  3/0.29  5/2.67 2/5.45 1/11.66 1/11.66
  5 -0.1829  0.4512  0.3605  5.9  3/2.47  4/2.67 2/4.38 1/12.88 1/13.92


 
and for dataset 2,
    80 residues left after pruning, divided into chains as follows:
A:  80
...
CC for partial structure against native data =  46.31 %
Estimated mean FOM and mapCC as a function of resolution
d    inf - 4.49 - 3.55 - 3.10 - 2.81 - 2.61 - 2.45 - 2.32 - 2.22 - 2.13 - 2.02
<FOM>  0.726  0.703  0.695  0.704  0.706  0.713  0.667  0.572  0.535  0.503
<mapCC> 0.850  0.863  0.857  0.899  0.900  0.908  0.866  0.805  0.828  0.814
N        719    721    725    719    713    736    755    722    673    705
Estimated mean FOM = 0.654  Pseudo-free CC = 67.40 %
Density (in map sigma units) at input heavy atom sites
  Site    x        y        z    occ*Z    density
    1  0.1613  0.5298  0.4706  16.0000    22.30
    2  0.1266  0.3414  0.5281  14.4576    17.03
    3  0.3453  0.2833  0.6078  11.1760    11.69
    4  0.0318  0.3665  0.5267  6.6512    8.45
    5  0.0499  0.3350  0.5280  5.8208    5.38
Site    x      y      z  h(sig) near old  near new
  1  0.1605  0.5316  0.4699  22.4  1/0.11  2/10.61 4/11.62 4/11.62 5/12.61
  2  0.1258  0.3407  0.5328  17.4  2/0.20  5/3.83 4/5.39 1/10.61 3/12.02
  3  0.3367  0.2831  0.6107  13.2  3/0.47  2/12.02 5/15.41 1/17.15 4/17.33
  4  0.0269  0.3630  0.5241  9.3  4/0.33  5/2.78 2/5.39 1/11.62 1/11.62
  5  0.0575  0.3206  0.5182  8.2  5/0.95  4/2.78 2/3.83 1/12.61 1/14.10


'''clearly indicating that the structure can be solved with each of the two datasets individually.'''
'''clearly indicating that the structure can be solved with each of the two datasets individually.'''


==Optimization of data reduction==
==Can we do better?==
 
===data reduction===
The safest way to optimize the data reduction is to look at external quality indicators. Internal R-factors, and even the correlation coefficient of the anomalous signal are of comparatively little value. A readily available external quality indicator is CC All/CC Weak as obtained by [[ccp4com:SHELX_C/D/E|SHELXD]].
The safest way to optimize the data reduction is to look at external quality indicators. Internal R-factors, and even the correlation coefficient of the anomalous signal are of comparatively little value. A readily available external quality indicator is CC All/CC Weak as obtained by [[ccp4com:SHELX_C/D/E|SHELXD]].


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[[Optimization]] does improve things as much as it often does: recycling of GXPARM.XDS to use as XPARM.XDS, and thus imposing the lattice symmetry in the geometry refinement in INTEGRATE. These findings my correspond to the fact that in P1 the angles do not refine to 90.0xx or 89.9xx degrees. In other words, the metric symmetry is not as well fulfilled as it should be. This results in fairly large deviations from the ideal P42 positions; the refinement of cell parameters in P1 partly compensates for this. I have however no idea why this deviation from metric symmetry could occur.  
[[Optimization]] does improve things as much as it often does: recycling of GXPARM.XDS to use as XPARM.XDS, and thus imposing the lattice symmetry in the geometry refinement in INTEGRATE. These findings my correspond to the fact that in P1 the angles do not refine to 90.0xx or 89.9xx degrees. In other words, the metric symmetry is not as well fulfilled as it should be. This results in fairly large deviations from the ideal P42 positions; the refinement of cell parameters in P1 partly compensates for this. I have however no idea why this deviation from metric symmetry could occur.  


==Optimization of structure solution==
===structure solution===


The resolution limit for SHELXD could be varied. For SHELXE, the solvent content could be varied, and the number of autobuilding cycles, and probably also the high resolution cutoff.
The resolution limit for SHELXD could be varied. For SHELXE, the solvent content could be varied, and the number of autobuilding cycles, and probably also the high resolution cutoff. Furthermore, it would be advantageous to "re-cycle" the file j.hat to j_fa.res, since the heavy-atom sites from SHELXE are more accurate than those from SHELXD, as the phases derived from the poly-Ala traces are quite good (compare the density columns of the two consecutive heavy-atom lists!).


==Limits==
==Limits==


With dataset 2, I tried to use 270 frames but could not solve the structure using the above SHELXC/D/E approach (not even with MAXIMUM_ERROR_OF_SPOT_POSITION=6.0). With 315 frames, it was possible.
With dataset 2, I tried to use 270 frames but could not solve the structure using the above SHELXC/D/E approach (not even with MAXIMUM_ERROR_OF_SPOT_POSITION=6.0). With 315 frames, it was possible.
Kay
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