# 2VB1

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This reports processing of triclinic hen egg-white lysozyme data @ 0.65Å resolution (PDB id 2VB1). Data (sweeps a to h, each comprising 60 to 360 frames of 72MB) were collected by Zbigniew Dauter at APS 19-ID and are available from here. Details of data collection, processing and refinement are published.

## XDS processing

1. use generate_XDS.INP to obtain a good starting point
2. edit XDS.INP and change/add the following:
ORGX=3130 ORGY=3040  ! for ADSC, header values are subject to interpretation; these values from visual inspection
! the following is for masking the beamstop shadow in sweeps c-d
UNTRUSTED_RECTANGLE=0 3189 2960 3087 ! use XDS-viewer of ADXV to find the values
! the following is for sweeps e-h
UNTRUSTED_RECTANGLE=1 3160 3000 3070
TRUSTED_REGION=0 1.5 ! we want the whole detector area
ROTATION_AXIS=-1 0 0 ! at this beamline the spindle goes backwards!
SILICON=34.812736 ! account for theta-dependant absorption in the CCD's phosphor. The correction is only
! significant for hi-res data; 34.812736=32*(value for silicon as printed to CORRECT.LP if SILICON= not given)
MAXIMUM_NUMBER_OF_PROCESSORS=4 ! for fast processing on a machine with many cores (e.g. for 16 cores)
MAXIMUM_NUMBER_OF_JOBS=6 ! "overcommit" the available cores but on the whole this produces results faster
SPACE_GROUP_NUMBER=1                   ! this is known
UNIT_CELL_CONSTANTS=  27.07 31.25 33.76 87.98 108.00 112.11  ! from 2vb1
FRIEDEL'S_LAW=TRUE  ! we're not concerned with the anomalous signal


Then, run "xds_par". It completes after about 5 minutes on a fast machine, and we may inspect (at least) IDXREF.LP and CORRECT.LP (see below), and use "XDS-viewer FRAME.cbf" to get a visual impression of the integration as it applies to the last frame. By inspecting IDXREF.LP, one should make sure that everything works as it should, i.e. that a large percentage of reflections was actually indexed nicely, e.g.:

...
63879 OUT OF   72321 SPOTS INDEXED.
...

***** DIFFRACTION PARAMETERS USED AT START OF INTEGRATION *****

REFINED VALUES OF DIFFRACTION PARAMETERS DERIVED FROM  63879 INDEXED SPOTS
REFINED PARAMETERS:   DISTANCE BEAM AXIS CELL ORIENTATION
STANDARD DEVIATION OF SPOT    POSITION (PIXELS)     0.53
STANDARD DEVIATION OF SPINDLE POSITION (DEGREES)    0.12



### Optimization

The main target of optimization is the asymptotic (i.e. best) I/sigma (ISa) (Diederichs (2010) Acta Cryst. D 66, 733-40) as printed out by CORRECT (and XSCALE). A higher ISa should mean better data.

However: ISa also rises if more reflections are thrown out as outliers ("misfits") so it is not considered to be optimization if just WFAC1 is reduced. Please note that the default WFAC1 is 1; this should result in the rejection of about 1% of observations. If you feel that 1% is too much then just increase WFAC1, to, say, 1.5 - that should result in rejection of less than (say) 0.1%. This will slightly increase completeness, but will reduce I/sigma and ISa, and increase R-factors.

The following quantities may be tested for their influence on ISa:

• copying GXPARM.XDS to XPARM.XDS
• including the information from the first integration pass into XDS.INP - just do "grep _E INTEGRATE.LP|tail -2" and get e.g.
BEAM_DIVERGENCE=   0.386  BEAM_DIVERGENCE_E.S.D.=   0.039
REFLECTING_RANGE=  0.669  REFLECTING_RANGE_E.S.D.=  0.096


copy these two lines into XDS.INP

• prevent refinement in INTEGRATE: REFINE(INTEGRATE)= !

## Example: sweep e

### XDS.INP; as generated by generate_XDS.INP

generate_XDS.INP "../../APS/19-ID/2vb1/p1lyso_e.0???.img"


Then include the changes detailed above, resulting in:

JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT
MAXIMUM_NUMBER_OF_PROCESSORS=4
MAXIMUM_NUMBER_OF_JOBS=6
ORGX= 3130 ORGY= 3040  ! check these values with adxv !
UNTRUSTED_RECTANGLE=1 3160 3000 3070  ! <xmin xmax ymin ymax> to mask shadow of beamstop; XDS-viewer to find out
DETECTOR_DISTANCE= 99.9954
OSCILLATION_RANGE= 0.500
X-RAY_WAVELENGTH=   0.6525486
NAME_TEMPLATE_OF_DATA_FRAMES=../../APS/19-ID/2vb1/p1lyso_e.0???.img
! 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=1                   ! 0 if unknown
UNIT_CELL_CONSTANTS= 27.07    31.25    33.76  87.98 108.00 112.11  ! PDB 2vb1
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
!
! 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 1.5  ! 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:
SENSOR_THICKNESS=0.01 SILICON=34.812736
NX= 6144 NY= 6144  QX= 0.051294  QY= 0.051294 ! 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



### CORRECT.LP 1st pass

STANDARD DEVIATION OF SPOT    POSITION (PIXELS)     0.87
STANDARD DEVIATION OF SPINDLE POSITION (DEGREES)    0.10
CRYSTAL MOSAICITY (DEGREES)     0.126

...

a        b          ISa
6.630E+00  1.091E-04   37.18

...

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

1.77        9195    4841      9501       51.0%       1.5%      1.5%     8708   48.74     2.1%     1.6%     0%   0.000       0
1.26       29991   15327     16721       91.7%       1.5%      1.6%    29328   45.26     2.1%     1.7%     0%   0.000       0
1.03       38643   19731     21636       91.2%       1.7%      1.7%    37824   38.67     2.5%     2.1%     0%   0.000       0
0.89       46156   23404     25561       91.6%       2.3%      2.4%    45504   27.56     3.3%     3.4%     0%   0.000       0
0.80       51509   26034     28868       90.2%       4.0%      4.0%    50950   17.55     5.6%     7.0%     0%   0.000       0
0.73       55989   28253     32034       88.2%       7.0%      6.8%    55472   10.98     9.8%    13.2%     0%   0.000       0
0.68       59733   30115     34776       86.6%      13.1%     13.0%    59236    6.08    18.6%    26.0%     0%   0.000       0
0.63       35385   18436     37367       49.3%      25.6%     26.9%    33898    2.99    36.3%    52.1%     0%   0.000       0
0.60        8991    4972     39725       12.5%      51.2%     56.9%     8038    1.34    72.4%   105.0%     0%   0.000       0
total      335592  171113    246189       69.5%       2.3%      2.4%   328958   19.58     3.3%     7.4%     0%   0.000       0

NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  343716
NUMBER OF REJECTED MISFITS                            8112
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS                  0
NUMBER OF ACCEPTED OBSERVATIONS                     335604
NUMBER OF UNIQUE ACCEPTED REFLECTIONS               171119


The number of "misfits" (rejections) is higher than expected (1 %). Either one considers the anomalous signal (of the 6 sulfurs) to be significant, or one simply increases WFAC1 from its default of 1, to (say) 1.2 .

### XDS.INP; optimized

Using the output of "grep _E INTEGRATE.LP|tail -2" edit XDS.INP to have

JOB= INTEGRATE CORRECT
BEAM_DIVERGENCE=   0.428  BEAM_DIVERGENCE_E.S.D.=   0.043
REFLECTING_RANGE=  0.880  REFLECTING_RANGE_E.S.D.=  0.126
...
REFINE(INTEGRATE)= !


Then "cp GXPARM.XDS XPARM.XDS", and then another round of "xds_par". Five minutes later, we get:

### CORRECT.LP optimization pass

This looks a little bit better - less standard deviation, higher ISa, better R-factors, less misfits:

STANDARD DEVIATION OF SPOT    POSITION (PIXELS)     0.83
STANDARD DEVIATION OF SPINDLE POSITION (DEGREES)    0.08
CRYSTAL MOSAICITY (DEGREES)     0.096

a        b          ISa
6.439E+00  1.076E-04   37.98

...

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

1.77        9149    4817      9501       50.7%       1.5%      1.5%     8664   49.75     2.1%     1.5%     0%   0.000       0
1.26       30049   15348     16723       91.8%       1.5%      1.6%    29402   46.26     2.1%     1.6%     0%   0.000       0
1.03       38920   19863     21637       91.8%       1.7%      1.7%    38114   39.61     2.4%     2.0%     0%   0.000       0
0.89       46381   23508     25562       92.0%       2.2%      2.3%    45746   28.39     3.1%     3.2%     0%   0.000       0
0.80       51605   26071     28868       90.3%       3.8%      3.8%    51068   18.21     5.3%     6.5%     0%   0.000       0
0.73       56126   28314     32041       88.4%       6.6%      6.4%    55624   11.45     9.3%    12.3%     0%   0.000       0
0.68       59735   30093     34771       86.5%      12.6%     12.3%    59284    6.34    17.8%    24.8%     0%   0.000       0
0.63       35754   18620     37370       49.8%      24.1%     25.5%    34268    3.11    34.1%    48.9%     0%   0.000       0
0.60        9180    5075     39730       12.8%      48.6%     54.3%     8210    1.40    68.7%   100.5%     0%   0.000       0
total      336899  171709    246203       69.7%       2.2%      2.3%   330380   20.14     3.2%     6.9%     0%   0.000       0

NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  344751
NUMBER OF REJECTED MISFITS                            7842
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS                  0
NUMBER OF ACCEPTED OBSERVATIONS                     336909
NUMBER OF UNIQUE ACCEPTED REFLECTIONS               171714


### further optimization

Another round of optimization again improves the R-factors and I/sigma at high resolution a bit, but it also increased the misfits back to 8200. At this point I decided to switch to FRIEDEL'S_LAW=FALSE, and the resulting table 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

1.77        9599    9023     19002       47.5%       1.5%      1.5%     1152   36.81     2.1%     1.6%     0%   0.000       0
1.26       31196   28239     33446       84.4%       1.4%      1.6%     5914   34.40     2.0%     1.6%     0%   0.000       0
1.03       40125   35205     43274       81.4%       1.7%      1.7%     9840   30.09     2.4%     2.0%     0%   0.000       0
0.89       46987   40188     51124       78.6%       2.3%      2.3%    13598   22.03     3.2%     3.4%     0%   0.000       0
0.80       52229   43723     57738       75.7%       3.9%      3.9%    17012   14.44     5.5%     6.6%     0%   0.000       0
0.73       56830   46674     64088       72.8%       7.1%      6.8%    20312    9.30    10.1%    13.2%     0%   0.000       0
0.68       60488   48814     69544       70.2%      13.9%     13.5%    23348    5.26    19.6%    27.1%     0%   0.000       0
0.63       36190   28598     74736       38.3%      28.2%     29.7%    15184    2.70    39.8%    57.3%     0%   0.000       0
0.60        9246    7246     79466        9.1%      57.8%     62.4%     4000    1.26    81.8%   122.0%     0%   0.000       0
total      342890  287710    492418       58.4%       2.8%      2.8%   110360   16.19     3.9%     9.9%     0%   0.000       0

NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  345355
NUMBER OF REJECTED MISFITS                            2448
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS                  0
NUMBER OF ACCEPTED OBSERVATIONS                     342907
NUMBER OF UNIQUE ACCEPTED REFLECTIONS               287724


Indeed this brings the number of misfits to well below 1%, and it does make some sense.

## XSCALE results

The same strategy as shown for sweep e was used for sweeps a-d and f-h. XSCALE.INP is:

SPACE_GROUP_NUMBER=    1
UNIT_CELL_CONSTANTS= 27.07 31.25 33.76 87.98 108.00 112.11 !  from 2vb1 PDB entry
! cellparm for a-h gives  27.083    31.269    33.773    87.978   107.998   112.133

OUTPUT_FILE=lys-xds.ahkl
FRIEDEL'S_LAW=TRUE
RESOLUTION_SHELLS=2.91 2.06 1.68 1.45 1.30 1.19 1.10 1.03 0.97 0.92 0.88 0.84 0.81 0.78 0.75 0.73 0.71 0.69 0.67 0.65

INPUT_FILE=../a/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65
INPUT_FILE=../b/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65
INPUT_FILE=../c/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65
INPUT_FILE=../d/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65
INPUT_FILE=../e/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65
INPUT_FILE=../f/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65
INPUT_FILE=../g/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65
INPUT_FILE=../h/XDS_ASCII.HKL
INCLUDE_RESOLUTION_RANGE=30 0.65


### XSCALE.LP tables

The error model is adjusted by XSCALE:

    a        b          ISa    ISa0   INPUT DATA SET
7.094E+00  1.294E-04   33.00   38.03 ../a/XDS_ASCII.HKL
7.476E+00  1.170E-04   33.81   38.95 ../b/XDS_ASCII.HKL
7.453E+00  1.598E-04   28.98   38.00 ../c/XDS_ASCII.HKL
6.539E+00  1.640E-04   30.54   39.08 ../d/XDS_ASCII.HKL
7.304E+00  1.342E-04   31.94   37.69 ../e/XDS_ASCII.HKL
8.201E+00  1.574E-04   27.83   35.58 ../f/XDS_ASCII.HKL
8.182E+00  1.759E-04   26.36   27.60 ../g/XDS_ASCII.HKL
7.717E+00  3.694E-04   18.73   21.93 ../h/XDS_ASCII.HKL


and there are about 1500 rejected reflections. It is reassuring to note that the error model works well; the ISa goes down toward sweep h probably because the crystal degrades. But see also the "a posterior remarks" below - sweep h is the one that is most affected by a shadow on the detector.

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

2.91       16170    2112      2147       98.4%       2.2%      2.4%    16157   78.96     2.5%     1.1%   -12%   0.741    2023
2.06       40349    3831      3856       99.4%       2.4%      2.7%    40345   84.89     2.6%     0.9%    -9%   0.764    3803
1.68       65329    5068      5087       99.6%       3.1%      3.2%    65321   83.77     3.3%     1.0%     0%   0.847    5020
1.45       73373    6147      6163       99.7%       3.2%      3.5%    73371   78.02     3.4%     1.0%     2%   0.842    6053
1.30       71196    6651      6657       99.9%       3.2%      3.5%    71196   71.07     3.4%     1.1%     4%   0.857    6503
1.19       74542    7287      7298       99.8%       3.2%      3.4%    74534   67.06     3.3%     1.2%     5%   0.854    7060
1.10       84918    8269      8278       99.9%       3.4%      3.7%    84891   63.24     3.6%     1.3%     7%   0.853    7988
1.03       87890    8584      8603       99.8%       4.1%      4.4%    87855   56.26     4.4%     1.5%     5%   0.818    8231
0.97       92917    9460      9465       99.9%       5.2%      5.6%    92894   48.90     5.5%     1.7%     4%   0.795    9010
0.92       83994    9911      9927       99.8%       5.7%      6.3%    83969   41.67     6.0%     2.0%     6%   0.787    9358
0.88       74100    9620      9621      100.0%       6.3%      7.1%    74082   35.74     6.7%     2.5%     4%   0.772    9040
0.84       81322   11511     11518       99.9%       6.9%      7.7%    81300   30.43     7.3%     3.3%     1%   0.760   10609
0.81       67539   10239     10247       99.9%       7.1%      7.7%    67518   25.96     7.7%     4.2%     2%   0.779    9364
0.78       73980   11807     11817       99.9%       7.1%      7.3%    73951   22.34     7.7%     5.3%     2%   0.799   10699
0.75       86111   13831     13839       99.9%       8.4%      8.6%    86076   18.77     9.2%     6.8%     2%   0.809   12496
0.73       64554   10481     10488       99.9%      10.3%     10.4%    64525   15.73    11.3%     8.2%     3%   0.815    9384
0.71       71891   11727     11741       99.9%      12.8%     13.0%    71844   12.95    14.0%    10.6%     3%   0.810   10436
0.69       80168   13157     13163      100.0%      16.6%     16.9%    80065   10.16    18.2%    14.1%     2%   0.799   11662
0.67       84431   14747     14766       99.9%      22.2%     22.7%    84231    7.44    24.4%    19.7%     3%   0.798   12520
0.65       61031   15592     16551       94.2%      27.6%     30.6%    60165    4.36    31.8%    33.1%     1%   0.723    9005
total     1435805  190032    191232       99.4%       3.1%      3.3%  1434290   33.42     3.3%     3.1%     3%   0.801  170264


If two more resolution shells are added, they look like -

    0.64       23276    7411      9155       81.0%      35.0%     40.6%    22324    2.90    41.7%    47.9%     3%   0.683    3204
0.63       18044    6488      9647       67.3%      42.2%     49.7%    16630    2.22    50.7%    60.9%    -5%   0.643    2437


So there is still useful signal beyond 0.65 A.

## Some a posteriori remarks

• For sweeps e-h one should use TRUSTED_REGION= 0 1.2 since that already gives 0.626 A in the corners.
• The first and last frames of sweeps g and h show a shadow in one corner of the detector. Nothing was done by me to exclude this shadow from processing (but one should do so at least if the resolution should be expanded beyond 0.65 A which the XSCALE statistics suggest to be possible).
One could experiment with MINIMUM_VALID_PIXEL_VALUE= 40 (or so) instead of 1 - I'd probably try that, but of course one does not want to exclude valid pixels so the result has to be carefully checked.
Anyway, there is no general facility in XDS to exclude bad areas of specific frames in a dataset; one needs to chop the dataset into parts and deal with each shadow separately.

## Comparison of data processing: published (2006) vs XDS results

 resolution (highest resolution range) observations unique reflections Multiplicity Completeness (%) R merge (%) mean I/sigma published (2006) 30-0.65Å (0.67-0.65Å) 1331953 (12764) 187165 (6353) 7.1 (2.7) 97.6 (67.3) 4.5 (18.4) 36.2 (4.2) XDS Version Dec 06, 2010 30-0.65Å (0.67-0.65Å) 1435805 (61031) 190032 (15592) 7.5 (3.9) 99.4 (94.2) 3.1 (27.6) 33.4 (4.4)

## Availability of data from XDS processing

I changed XSCALE.INP to have

!FRIEDEL'S_LAW=TRUE  ! by commenting it out XSCALE will use FRIEDEL'S_LAW=FALSE
!                      since this is how the data were processed
RESOLUTION_SHELLS=2.91 2.06 1.68 1.45 1.30 1.19 1.10 1.03 0.97 0.92 0.88 0.84 0.80 0.76 0.73 0.70 0.67 0.65 0.64 0.63


and ran XSCALE again, to get a file with reflections to 0.63 A.

Conversion to other program systems is performed with XDSCONV. XDSCONV.INP for producing a MTZ file with intensities and anomalous signal is:

INPUT_FILE= lys-xds.ahkl
OUTPUT_FILE=temp.hkl CCP4_I


After running xdsconv, I cut-and-paste the screen output:

f2mtz HKLOUT temp.mtz<F2MTZ.INP