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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>
<pre>
JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
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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
indicating at most tetragonal symmetry, shortly after this calculates R-factors for these lattices:
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
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  NUMBER OF UNIQUE ACCEPTED REFLECTIONS                13784
  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 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.
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.
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===dataset 2===
===dataset 2===
This works exactly the same way as dataset 1. The table in CORRECT.LP is
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.
       NOTE:      Friedel pairs are treated as different reflections.
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  NUMBER OF UNIQUE ACCEPTED REFLECTIONS                13738
  NUMBER OF UNIQUE ACCEPTED REFLECTIONS                13738


Dataset 2 is definitively better than dataset 1.
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 (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 (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
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  shelxd j_fa
  shelxd j_fa


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 .  
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.  


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 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  
and the the inverse hand:
and the inverse hand:
  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


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==Can we do better?==
==Can we do better?==
===data reduction===
===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]], and the percentage of successful trials.


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. In general, it makes sense to use MAXIMUM_ERROR_OF_SPOT_POSITION= (at least 3 times the STANDARD DEVIATION OF SPOT POSITION (PIXELS)) 
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.


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!
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.


[[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.
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!


===structure solution===
===structure solution===
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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!).
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==
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
 
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.
 
==Exploring the limits==
 
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!
 
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!'''
 
==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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