2QVO.xds

XDS data reduction

dataset 1

Using "generate_XDS.INP ../../APS/22-ID/2qvo/ACA10_AF1382_1.0???" we obtain:

JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
ORGX= 1996.00 ORGY= 2028.00  ! check these values with adxv !
DETECTOR_DISTANCE= 125.000
OSCILLATION_RANGE= 1.000
X-RAY_WAVELENGTH= 1.90000
NAME_TEMPLATE_OF_DATA_FRAMES=../../APS/22-ID/2qvo/ACA10_AF1382_1.0???
! REFERENCE_DATA_SET=xxx/XDS_ASCII.HKL ! e.g. to ensure consistent indexing  
DATA_RANGE=1 360
SPOT_RANGE=1 180
! BACKGROUND_RANGE=1 10 ! rather use defaults (first 5 degree of rotation)

SPACE_GROUP_NUMBER=0                   ! 0 if unknown
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

FRIEDEL'S_LAW=FALSE     ! This acts only on the CORRECT step
! If the anom signal turns out to be, or is known to be, very low or absent,
! use FRIEDEL'S_LAW=TRUE instead (or comment out the line); re-run CORRECT

! remove the "!" in the following line:
! STRICT_ABSORPTION_CORRECTION=TRUE
! if the anomalous signal is strong: in that case, in CORRECT.LP the three
! "CHI^2-VALUE OF FIT OF CORRECTION FACTORS" values are significantly> 1, e.g. 1.5
!
! exclude (mask) untrusted areas of detector, e.g. beamstop shadow :
! UNTRUSTED_RECTANGLE= 1800 1950 2100 2150 ! x-min x-max y-min y-max ! repeat
! UNTRUSTED_ELLIPSE= 2034 2070 1850 2240 ! x-min x-max y-min y-max ! if needed
!
! parameters with changes wrt default values:
TRUSTED_REGION=0.00 1.2  ! partially use corners of detectors; 1.41421=full use
VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS=7000. 30000. ! often 8000 is ok
MINIMUM_ZETA=0.05        ! integrate close to the Lorentz zone; 0.15 is default
STRONG_PIXEL=6           ! COLSPOT: only use strong reflections (default is 3)
MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT=3 ! default of 6 is sometimes too high
REFINE(INTEGRATE)=CELL BEAM ORIENTATION ! AXIS DISTANCE 

! parameters specifically for this detector and beamline:
DETECTOR= CCDCHESS MINIMUM_VALID_PIXEL_VALUE= 1 OVERLOAD= 65500
NX= 4096 NY= 4096  QX= .0732420000  QY= .0732420000 ! to make CORRECT happy if frames are unavailable
DIRECTION_OF_DETECTOR_X-AXIS=1 0 0
DIRECTION_OF_DETECTOR_Y-AXIS=0 1 0
INCIDENT_BEAM_DIRECTION=0 0 1
ROTATION_AXIS=1 0 0    ! at e.g. SERCAT ID-22 this needs to be -1 0 0
FRACTION_OF_POLARIZATION=0.98   ! better value is provided by beamline staff!
POLARIZATION_PLANE_NORMAL=0 1 0

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

 LATTICE-  BRAVAIS-   QUALITY  UNIT CELL CONSTANTS (ANGSTROEM & DEGREES)    REINDEXING TRANSFORMATION
CHARACTER  LATTICE     OF FIT      a      b      c   alpha  beta gamma

*  44        aP          0.0      41.2   53.5   53.5  90.3  90.1  90.1   -1  0  0  0  0  1  0  0  0  0 -1  0
*  31        aP          0.8      41.2   53.5   53.5  89.7  90.1  89.9    1  0  0  0  0  1  0  0  0  0  1  0
*  25        mC          1.4      75.4   75.8   41.2  90.0  90.1  90.0    0  1 -1  0  0 -1 -1  0 -1  0  0  0
*  35        mP          1.8      53.5   41.2   53.5  90.1  90.3  90.1    0 -1  0  0  1  0  0  0  0  0  1  0
*  23        oC          3.1      75.4   75.8   41.2  90.0  90.1  90.0    0  1 -1  0  0 -1 -1  0 -1  0  0  0
*  20        mC          3.9      75.8   75.4   41.2  90.1  90.0  90.0    0  1  1  0  0  1 -1  0 -1  0  0  0
*  34        mP          5.1      41.2   53.5   53.5  90.3  90.1  90.1    1  0  0  0  0  0  1  0  0 -1  0  0
*  33        mP          5.3      41.2   53.5   53.5  90.3  90.1  90.1   -1  0  0  0  0  1  0  0  0  0 -1  0
*  32        oP          6.1      41.2   53.5   53.5  90.3  90.1  90.1   -1  0  0  0  0  1  0  0  0  0 -1  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

indicating at most tetragonal symmetry, shortly after this calculates R-factors for these lattices:

SPACE-GROUP         UNIT CELL CONSTANTS            UNIQUE   Rmeas  COMPARED  LATTICE-
  NUMBER      a      b      c   alpha beta gamma                            CHARACTER

      5      75.8   75.4   41.2  90.0  90.0  90.0     900    40.8     5882    20 mC
  *  75      53.5   53.5   41.2  90.0  90.0  90.0     469     8.4     6313    21 tP
     89      53.5   53.5   41.2  90.0  90.0  90.0     282    39.2     6500    21 tP
     21      75.4   75.8   41.2  90.0  90.0  90.0     506    39.8     6276    23 oC
      5      75.4   75.8   41.2  90.0  90.1  90.0     901    40.7     5881    25 mC
      1      41.2   53.5   53.5  89.7  90.1  89.9    1699     8.2     5083    31 aP
     16      41.2   53.5   53.5  90.0  90.0  90.0     521    39.8     6261    32 oP
      3      53.5   41.2   53.5  90.0  90.3  90.0     931     8.2     5851    35 mP
      3      41.2   53.5   53.5  90.0  90.1  90.0     918    40.7     5864    33 mP
      3      41.2   53.5   53.5  90.0  90.1  90.0     918    40.9     5864    34 mP
      1      41.2   53.5   53.5  90.3  90.1  90.1    1699     8.2     5083    44 aP

thus suggesting spacegroup #75 but we should know that this does not take screw axes into account. Therefore we use "pointless xdsin XDS_ASCII.HKL" and are told that this is actually spacegroup P4_2 (# 77). Alternatively, we could have inspected the list further down in CORRECT.LP:

  REFLECTIONS OF TYPE H,0,0  0,K,0  0,0,L OR EXPECTED TO BE ABSENT (*)
  --------------------------------------------------------------------

  H    K    L  RESOLUTION  INTENSITY     SIGMA    INTENSITY/SIGMA  #OBSERVED

   0    0    1    41.248   0.8487E+01  0.1339E+01         6.34           4 
   0    0    3    13.749  -0.7977E-03  0.3786E+01         0.00           4 
   0    0    4    10.312   0.1305E+06  0.4660E+04        27.99           1 
   0    0    5     8.250   0.1318E+01  0.6316E+01         0.21           4 
   0    0    6     6.875   0.2939E+05  0.5284E+03        55.61           4 
   0    0    7     5.893   0.5439E+01  0.9235E+01         0.59           4 
   0    0    8     5.156   0.1298E+05  0.2371E+03        54.73           4 
   0    0    9     4.583   0.3308E+02  0.1327E+02         2.49           4 
   0    0   10     4.125   0.3809E+05  0.6867E+03        55.47           4 
   0    0   11     3.750  -0.1987E+02  0.1767E+02        -1.12           4 
   0    0   12     3.437   0.5539E+04  0.1097E+03        50.48           4 
   0    0   13     3.173   0.2144E+01  0.2071E+02         0.10           4 
   0    0   14     2.946   0.2717E+04  0.6252E+02        43.46           4 
   0    0   15     2.750   0.1350E+02  0.2482E+02         0.54           4 
   0    0   16     2.578   0.1178E+04  0.4383E+02        26.88           4 
   0    0   17     2.426  -0.7420E+01  0.3549E+02        -0.21           4 
   0    0   18     2.292   0.4104E+03  0.4681E+02         8.77           4 

and realize that this also tells us that the spacegroup is 77, not 75.

After his comes the table that tells us the quality of our data:

      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        4189     556       560       99.3%       2.4%      2.7%     4187   66.74     2.6%     1.1%    74%   1.841     247
    4.31        7575    1008      1008      100.0%       2.6%      2.9%     7575   62.90     2.8%     1.2%    62%   1.463     473
    3.53        9468    1283      1283      100.0%       3.4%      3.2%     9468   53.37     3.6%     1.7%    41%   1.200     612
    3.06       11364    1540      1540      100.0%       5.1%      4.7%    11364   34.45     5.5%     3.1%    17%   0.995     739
    2.74       12628    1695      1695      100.0%      10.2%     10.4%    12628   17.09    11.0%     7.9%     2%   0.797     819
    2.50       14121    1916      1916      100.0%      21.5%     23.1%    14121    8.42    23.1%    17.1%    -4%   0.691     926
    2.31       15155    2079      2079      100.0%      46.6%     50.5%    15155    3.92    50.2%    38.6%    -1%   0.734    1010
    2.16       12185    2104      2228       94.4%     113.3%    117.0%    12178    1.44   124.7%   119.0%     5%   0.753    1018
    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


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).

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

JOB=CORRECT  ! not XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
SPACE_GROUP_NUMBER=   77
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).

Following this, we create XDSCONV.INP with the lines

SPACE_GROUP_NUMBER=   77  ! can leave out if CORRECT already ran in #77
UNIT_CELL_CONSTANTS=  53.03   53.03  40.97 90 90 90 ! same here
INPUT_FILE=XDS_ASCII.HKL
OUTPUT_FILE=temp.hkl CCP4

and run "xdsconv", and then

f2mtz HKLOUT temp.mtz<F2MTZ.INP
cad HKLIN1 temp.mtz HKLOUT output_file_name.mtz<<EOF
LABIN FILE 1 ALL
END
EOF

which gives us output_file_name.mtz, which we rename to xds-2ovo-1-F.mtz. Similarly, using

OUTPUT_FILE=temp.hkl CCP4_I

we end up with a MTZ file with intensities, which we rename to xds-2ovo-1-I.mtz.

dataset 2

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

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 use a script to generate XDSCONV.INP (I used MERGE=TRUE, sometimes the results are better that way), run xdsconv and SHELXC.

#!/bin/csh -f
 
cat > XDSCONV.INP <<end
INPUT_FILE=../XDS_ASCII.HKL
OUTPUT_FILE=temp.hkl SHELX
MERGE=TRUE
FRIEDEL'S_LAW=FALSE
end
 
xdsconv 
 
shelxc j <<end
SAD   temp.hkl
CELL 53.03 53.03 40.97 90 90 90
SPAG P42
MAXM 2
end

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 hkl2map would write):

cat > j_fa.ins <<end
TITL j_fa.ins SAD in P42
CELL  0.98000  53.03   53.03  40.97   90.00   90.00   90.00
LATT  -1
SYMM -Y, X, 1/2+Z
SYMM -X, -Y, Z
SYMM Y, -X, 1/2+Z
SFAC S
UNIT   128
SHEL 999 3.0
FIND 3
NTRY 100
MIND -1.0 2.2
ESEL 1.3
TEST 0 99
SEED 1
PATS
HKLF 3
END
end

and then

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 .

Next we run G. Sheldrick's beta-Version of SHELXE Version 2011/1:

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

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

  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.

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 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 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!

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.

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. 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 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.