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This is an example of S-SAD structure solution (PDB id [http://www.rcsb.org/pdb/explore.do?structureId=2QVO 2QVO]), a 95-residue protein used by James Tucker Swindell II to establish optimized procedures for data reduction. The data available to solve the structure are two runs of 360° collected at a wavelength of 1.9Å.
==XDS data reduction==
==XDS data reduction==
In the course of writing this up, it turned out that it was not necessary to scale the two datasets together, using [[XSCALE]], because the structure can be solved from any of the two, separately. But, of course, structure solution would be easier when merging the data (try for yourself!).


===dataset 1===
===dataset 1===


Using "generate_XDS.INP ../../APS/22-ID/2qvo/ACA10_AF1382_1.0???" we obtain:
Using [[generate_XDS.INP]] "../../APS/22-ID/2qvo/ACA10_AF1382_1.0???" we obtain:
<pre>
JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
ORGX= 1996.00 ORGY= 2028.00  ! check these values with adxv !
ORGX= 1996.00 ORGY= 2028.00  ! check these values with adxv !
Line 18: Line 23:
UNIT_CELL_CONSTANTS= 70 80 90 90 90 90 ! put correct values if known
UNIT_CELL_CONSTANTS= 70 80 90 90 90 90 ! put correct values if known
INCLUDE_RESOLUTION_RANGE=50 0  ! after CORRECT, insert high resol limit; re-run CORRECT
INCLUDE_RESOLUTION_RANGE=50 0  ! after CORRECT, insert high resol limit; re-run CORRECT


FRIEDEL'S_LAW=FALSE    ! This acts only on the CORRECT step
FRIEDEL'S_LAW=FALSE    ! This acts only on the CORRECT step
Line 50: Line 54:
FRACTION_OF_POLARIZATION=0.98  ! better value is provided by beamline staff!
FRACTION_OF_POLARIZATION=0.98  ! better value is provided by beamline staff!
POLARIZATION_PLANE_NORMAL=0 1 0
POLARIZATION_PLANE_NORMAL=0 1 0
</pre>


Now we run xds_par. This runs to completion. We should at least inspect, using XDS-Viewer, the file FRAME.cbf since this shows us the last frame of the dataset, with boxes superimposed which correspond to the expected locations of reflections.  
Now we run "xds_par". This runs to completion. We should at least inspect, using [[XDS-Viewer]], the file FRAME.cbf since this shows us the last frame of the dataset, with boxes superimposed which correspond to the expected locations of reflections.  


The automatic spacegroup determination (CORRECT.LP) comes up with
The automatic spacegroup determination (CORRECT.LP) comes up with
Line 68: Line 73:
  *  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. Below this table, CORRECT calculates R-factors for each of the lattices whose metric symmetry is compatible with the cell of the crystal (marked by * in the table above):
  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
Line 110: Line 115:


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.
   
   
Line 126: Line 132:
     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
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).
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 Å (which is where 30% would be, in the "Anomal Corr" column), and the useful resolution goes to 2.16 Å, 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
Line 152: Line 168:


===dataset 2===
===dataset 2===
This works exactly the same way as dataset 1.
This works exactly the same way as dataset 1. The geometry refinement is surprisingly bad:
REFINED PARAMETERS:  DISTANCE BEAM ORIENTATION CELL AXIS                 
USING  49218 INDEXED SPOTS
STANDARD DEVIATION OF SPOT    POSITION (PIXELS)    1.78
STANDARD DEVIATION OF SPINDLE POSITION (DEGREES)    0.15
CRYSTAL MOSAICITY (DEGREES)    0.218
DIRECT BEAM COORDINATES (REC. ANGSTROEM)  0.002198 -0.000174  0.526311
DETECTOR COORDINATES (PIXELS) OF DIRECT BEAM    1991.28  2027.42
DETECTOR ORIGIN (PIXELS) AT                    1984.09  2027.99
CRYSTAL TO DETECTOR DISTANCE (mm)      126.03
LAB COORDINATES OF DETECTOR X-AXIS  1.000000  0.000000  0.000000
LAB COORDINATES OF DETECTOR Y-AXIS  0.000000  1.000000  0.000000
LAB COORDINATES OF ROTATION AXIS  0.999979  0.002580 -0.006016
COORDINATES OF UNIT CELL A-AXIS  -31.728    -7.177  -42.595
COORDINATES OF UNIT CELL B-AXIS    40.575    13.173  -32.443
COORDINATES OF UNIT CELL C-AXIS    11.394  -39.576    -1.819
REC. CELL PARAMETERS  0.018658  0.018658  0.024258  90.000  90.000  90.000
UNIT CELL PARAMETERS    53.595    53.595    41.224  90.000  90.000  90.000
E.S.D. OF CELL PARAMETERS  1.0E-02 1.0E-02 1.7E-02 0.0E+00 0.0E+00 0.0E+00
SPACE GROUP NUMBER    75
with its large "STANDARD DEVIATION OF SPOT POSITION (PIXELS)" which may indicate a slipping crystal, or changing cell parameters due to radiation damage. However no indication of any of this is found in the repeated refinements listed in INTEGRATE.LP, so we do not know what to attribute this problem to!
 
The main 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. Note that the number of misfits is more than 2.5% whereas one should expect about 1% (with WFAC1=1).


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


This is done in a subdirectory of the XDS data reduction directory. 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 (of dataset "1" or "2"). Here, we use a script to generate XDSCONV.INP (I used MERGE=TRUE, sometimes the results are better that way; update Sep 2011: the [[ccp4com:SHELX_C/D/E#Obtaining_the_SHELX_programs|beta-test version of SHELXC]] fixes this problem, so MERGE=FALSE would be preferable since it gives more statistics output), run [[XDSCONV|xdsconv]] and [[ccp4com:SHELX_C/D/E|SHELXC]].
<pre>
<pre>
#!/bin/csh -f
#!/bin/csh -f
Line 171: Line 235:
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:
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>
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
Line 197: Line 262:
END
END
end
end
   
</pre>
shelxd j_fa
and then
  shelxd j_fa
 
The "FIND 3" needs a comment: the sequence has 4 Met and 1 Cys, but we don't expect to find the N-terminal Met. Since SHELXD always searches for more atoms than specified, we might as well tell it to try and locate 3 sulfurs.
 
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.


This gives best CC All/Weak of 35.61 / 26.03 for dataset 2, and best CC All/Weak of 36.74 / 21.55 for dataset 1.
Next we run G. Sheldrick's beta-Version of [[ccp4com:SHELX_C/D/E|SHELXE]] Version 2011/1:


Next we run G. Sheldrick's beta-Version of [[ccp4com:SHELX_C/D/E|SHELXE]] Version 2009/4:
shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b
and the inverse hand:
shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b -i


shelxe.beta j j_fa -a6 -q -h -s0.55 -m20 -b
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 2, I get
Some important lines in the output: for dataset 1, I get
    79 residues left after pruning, divided into chains as follows:
  78 residues left after pruning, divided into chains as follows:
  A:  20  B: 22  C: 37
  A:  78
  CC for partial structure against native data = 36.54 %
   
   
CC for partial structure against native data =  50.42 %
  ...
  ...
   <wt> = 0.300, Contrast = 0.731, Connect. = 0.817 for dens.mod. cycle 20
  ...
Estimated mean FOM and mapCC as a function of resolution
  Estimated mean FOM = 0.659   Pseudo-free CC = 68.71 %
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


for dataset 1, I get
and for dataset 2,
     80 residues left after pruning, divided into chains as follows:
     80 residues left after pruning, divided into chains as follows:
  A:  23  B:  57
  A:  80
 
   
CC for partial structure against native data = 45.79 %
  ...
  ...
  <wt> = 0.300, Contrast = 0.711, Connect. = 0.812 for dens.mod. cycle 20
  ...
CC for partial structure against native data =  46.31 %
  Estimated mean FOM = 0.611   Pseudo-free CC = 63.70 %
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.'''


==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]], and the percentage of successful trials.


For completeness, we run the inverse hand:
I tried a number of possibilities:
* [[Optimization]] by "re-cycling" GXPARM.XDS to XPARM.XDS and re-running INTEGRATE, coupled with REFINE(INTEGRATE)= ! (empty list) and specifying BEAM_DIVERGENCE_E.S.D. and similar parameters as obtained from INTEGRATE.LP: this quite often helps to improve geometry a bit but had no clear effect here.
* STRICT_ABSORPTION_CORRECTION=TRUE - this is useful if the chi^2 -values of the three scaling steps in CORRECT.LP are 1.5 and higher which is not the case here. Consequently this also had no clear effect.
* increasing MAXIMUM_ERROR_OF_SPOT_POSITION from its default of 3 to ( 3 * STANDARD DEVIATION OF SPOT POSITION (PIXELS)) which would mean increasing to 5 here: no clear effect.
* increasing WFAC1 : this was suggested by the number of misfits which is clearly higher than the usual 1 % of observations. WFAC1=1.5 has indeed a very positive effect on SHELXD: for dataset 1, the best CC All/Weak becomes '''44.93 / 22.82''' (dataset 2: '''48.11 / 27.78'''), and the number of successful trials goes from about 60% to 91% (dataset 2: 94%).''' One should note that all internal quality indicators get worse when increasing WFAC1 - but the external ones got significant better!''' The number of misfits with WFAC1=1.5 dropped to 196 / 436 for datasets 1 and 2, respectively.
* MERGE=FALSE vs MERGE=TRUE in XDSCONV.INP: after finding out about WFAC1 I tried MERGE=FALSE (the default !) and it turned out to be a bit better - best CC All/Weak '''48.66 / 28.05''' for dataset 2. On the other hand, the number of successful trials went down to 77% (from 94%). This result is somewhat difficult to interpret, but I like MERGE=TRUE better.


shelxe.beta j j_fa -a6 -q -h -s0.55 -m20 -b -i
We may thus conclude that in this case the rejection of misfits beyond the target value of 1% reduces data quality significantly. In (other) desperate cases, if no successful trials are made by SHELXD it may be worth to always try WFAC1=1.5 provided the number of misfits is high.


but of course this gives much worse statistics.
We also learn that it's usually ''not'' going to help much to deviate from the defaults (MERGE=, MAXIMUM_ERROR_OF_SPOT_POSITION=, STRICT_ABSORPTION_CORRECTION=) unless there is a clear reason (high number of misfits) to!


==Optimization of data reduction==
===structure solution===


The only safe 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 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!).


WFAC1 was already discussed above. Another candidate for optimization is MAXIMUM_ERROR_OF_SPOT_POSITION. By default this is 3.0 . In the case of these data, this default appears to be too small, because the STANDARD DEVIATION OF SPOT   POSITION (PIXELS) (as reported by IDXREF, INTEGRATE and CORRECT after refinement) is quite high (1.5 and more). This prevents XDS from using all the reflections for geometry refinement.
With the optimally-reduced dataset 2, I get from SHELXE:
Density (in map sigma units) at input heavy atom sites
  Site    x        y        z    occ*Z    density
    1  0.3361  0.9695  0.9827  16.0000    24.15
    2  0.3708  1.1540  1.0380  14.5216    17.48
    3   0.1576  1.2210  1.1222  9.2848    12.60
    4  0.4807  1.1304  1.0314  7.2224    8.95
    5  0.4539  1.1750  1.0368  6.6224    7.26
Site   x      y      z  h(sig) near old  near new
  1  0.3380  0.9687  0.9828  24.3  1/0.11  6/2.40 2/10.33 4/11.42 4/11.81
  2  0.3732  1.1546  1.0426  18.1  2/0.23  5/4.00 4/5.67 6/9.92 1/10.33
  3  0.1637  1.2180  1.1226  13.5  3/0.36  2/12.06 5/15.47 6/15.97 1/17.12
  4  0.4784  1.1371  1.0333  9.3  4/0.38  5/2.89 2/5.67 1/11.42 1/11.81
  5  0.4439  1.1791  1.0300  9.0  5/0.64  4/2.89 2/4.00 6/12.54 1/12.64
  6  0.3273  0.9734  1.0393  -5.9  1/2.38  1/2.40 2/9.92 4/11.82 4/11.86


I found that MAXIMUM_ERROR_OF_SPOT_POSITION=6.0 significantly improved the internal statistics (mostly the r-factors, but not so much the correlation coefficient of the anom signal), and improved CC All/CC Weak indicators (to more than 40). SHELXE then produces significantly better and more complete models. Try for yourself!
so the density is better, but not much. Furthermore, we note in passing that the number of anomalous scatterers (5) matches the sum of 4 Met and 1 Cys in the sequence.


One thing I noticed that if I specify the known spacegroup in IDXREF then the results are worse than if the integration is performed in P1. Likewise, [[optimization]] did not work: 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.
==Exploring the limits==


==Optimization of structure solution==
With dataset 2, I tried to use the first 270 frames and could indeed solve the structure using the above SHELXC/D/E approach (with WFAC1=1.5) - 85 residues in a single chain, with "CC for partial structure against native data = 47.51 %". It should be mentioned that I also tried this in November 2009, and it didn't work with the version of XDS available then!


There are some parameters in the SHELXC/D/E approach above that could be optimized as well: first of all, MERGE=TRUE in XDSCONV.INP turned later out to be the wrong choice (using the default MERGE=FALSE turns out to give a model with 85 consecutive residues for dataset 1). Then of course, the resolution limit for SHELXD could be varied, and the solvent content for SHELXE. For SHELXE in particular, many things could be tried.
With 180 frames, it was possible to get a complete model by (twice) re-cycling the j.hat file to j_fa.res. '''This means that the structure can be automatically solved just from the first 180 frames of dataset 2!'''


==Limits==
==Availability==
* [https://{{SERVERNAME}}/pub/xds-datared/2qvo/xds-2qvo-1-1_360-F.mtz] - amplitudes  for frames 1-360 of dataset 1.
* [https://{{SERVERNAME}}/pub/xds-datared/2qvo/xds-2qvo-1-1_360-I.mtz] - intensities for frames 1-360 of dataset 1.
* [https://{{SERVERNAME}}/pub/xds-datared/2qvo/xds-2qvo-2-1_180-F.mtz] - amplitudes  for frames 1-180 of dataset 2.
* [https://{{SERVERNAME}}/pub/xds-datared/2qvo/xds-2qvo-2-1_180-I.mtz] - intensities for frames 1-180 of dataset 2.
* [https://{{SERVERNAME}}/pub/xds-datared/2qvo/xds-2qvo-2-1_360-F.mtz] - amplitudes  for frames 1-360 of dataset 2.
* [https://{{SERVERNAME}}/pub/xds-datared/2qvo/xds-2qvo-2-1_360-I.mtz] - intensities for frames 1-360 of dataset 2.


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.
As you can see, all these files are in the same directory [https://{{SERVERNAME}}/pub/xds-datared/2qvo/]. I put there the XDS_ASCII.HKL files and SHELXD/SHELXE result files as well.
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