Trypsin ACA2014

From XDSwiki

7200 frames collected (and 3600 frames provided) on a Pilatus 2M detector at Photon Factory BL-1A. What is special about these data:

  • shutterless and fast (0.1°/frame, 0.1 sec/frame)
  • low energy (2.7 A wavelength)
  • cryoprotectant surrounding the crystal was partially removed to reduce X-ray absorption.

We start processing by creating an empty directory, and changing to this directory. This can be conveniently done using XDSGUI / Projects / navigate to the directory with the Trypsin frames / create empty folder.

DEFPIX (determination of valid detector area for integration)

In DEFPIX, the trusted pixels (used for INTEGRATE) are determined. To exclude beam stop shadow etc., the background value of each pixel is compared with resolution-averaged background velue. Usually default parameter works well, but sometimes it needs adjustment like this dataset. Check BKGPIX.cbf, the initial background table for INTEGRATE, where untrusted pixels are marked by -3.

BKGPIX.cbf (colored)
ABS.cbf

Open BKGPIX.cbf with adxv or xds-viewer. In this figure, negative pixels (untrusted pixels) are colored yellow. Apparently, valid area are masked as well as beam stop shadow.

Check ABS.cbf generated in DEFPIX. The pixel value is the ratio between the background and the mean background of the same resolution, multiplied by 10000. If the pixel value is outside VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS= (default of generate_XDS.INP rev. 0.44 is "6000 30000"), the pixel is marked as untrusted one. The wrongly masked area have the value from 3000 to 6000 here. Edit XDS.INP and change the line to VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS= 3000 30000, and then run DEFPIX again.

Check the revised BKGPIX.cbf - looks alright?

Revised BKGPIX.cbf (colored)


How Low (In Frames) Can You Go?

With JOB= CORRECT DATA_RANGE= 1 3600,

 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  CC(1/2)  Anomal  SigAno   Nano
   LIMIT     OBSERVED  UNIQUE  POSSIBLE     OF DATA   observed  expected                                      Corr

     7.03        4354     612       616       99.4%       4.2%      3.8%     4354   45.53      4.5%    99.8*    93*   4.398     226
     5.01        7329    1082      1087       99.5%       4.1%      3.9%     7329   43.28      4.5%    99.9*    91*   3.682     463
     4.10        8684    1350      1375       98.2%       4.0%      3.8%     8679   43.72      4.3%    99.8*    82*   2.411     601
     3.55       10993    1606      1639       98.0%       3.6%      3.9%    10989   44.34      3.9%    99.9*    77*   2.011     726
     3.18       11841    1813      1869       97.0%       3.8%      4.0%    11836   40.35      4.1%    99.8*    71*   2.023     829
     2.90       12753    1945      2048       95.0%       3.8%      4.4%    12750   35.85      4.1%    99.9*    76*   2.057     900
     2.69       13877    2083      2214       94.1%       4.1%      4.9%    13875   32.19      4.4%    99.8*    75*   1.918     971
     2.52       14118    2199      2409       91.3%       4.9%      5.3%    14104   28.22      5.3%    99.7*    74*   1.817    1019
     2.37        6456    1606      2539       63.3%       5.6%      5.7%     6271   19.22      6.4%    99.5*    62*   1.446     653
    total       90405   14296     15796       90.5%       4.0%      4.2%    90187   35.52      4.3%    99.9*    79*   2.159    6388

With 3,600 frames (360 degrees), phasing is straightforward.