3CSL

HasA/R (PDB id 3CSL) is a complex of a 22-stranded beta-barrel outer membrane protein (HAsR, 865 residues), its hemophore (HasA, 206 residues), and heme. The structure and its biological implications are described in "Heme uptake across the outer membrane as revealed by crystal structures of the receptor-hemophore complex" (Krieg, S., Huché, F., Diederichs, K., Izadi-Pruneyre, N., Lecroisey, A., Wandersman, C., Delepelaire, P., Welte, W. (2009), Proc. Nat. Acad. Sci. Vol. 106 pp. 1045-1050.)

3-wl SeMet-MAD data were collected at beamline X06SA of the SLS in November 2006 on a MarCCD detector. HasA/R crystallizes in spacegroup F222; cell parameters are a=157Å, b=163Å, c=596Å. There are 2 complexes per ASU. Data to about 3.0Å could be collected from this crystal, but the anomalous data are useful to about 5Å only. The ordered part of HasR has residues 112-865 and harbours 9 SeMet residues. The ordered part of HasA has 173 residues, one of which is SeMet - but that is mostly disordered.

These MAD data, giving a structure with an average B of 100 Å2, constitute a project that is challenging for humans, and currently too difficult for automatic methods of structure solution and model building. The deposited 3CSL structure was not obtained from these MAD data alone, but the model was actually refined against slightly better (2.7Å) data collected on a native crystal at the ESRF.

XDS data reduction of high-remote, peak and inflection

The script generate_XDS.INP may be used to get a suitable first XDS.INP file for each of the three wavelengths. Unfortunately the beamline software did not put the correct X and Y position of the direct beam into the header. So you will have to find this yourself, using adxv or XDS-viewer. Or just use:

ORGX= 1536 ORGY= 1520 

The other thing that you might want to try yourself, or just fill in, is

VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS=8000. 30000. ! often 8000 is ok

instead of the the default (7000. 30000.). This results in a good mask for the beamstop shadow.

It turns out that the spot shapes are actually so irregular that XDS stops after the IDXREF step, with a long warning message. This is because it cannot index (within default error margins) enough reflections (50% is the cutoff). When that occurs, one simply continues with the step after IDXREF:

JOBS= DEFPIX INTEGRATE CORRECT 

Other than that, the three MAD wavelengths can be processed once with default parameters, as written into XDS.INP by generate_XDS.INP. This data reduction therefore proceeds in spacegroup P1, but the correct spacegroup (22) is identified by CORRECT.

Optimization: after this first data reduction pass, I use the "post-refined" geometric parameters, and the correct spacegroup (as given in CORRECT.LP, and written to GXPARM.XDS), for a second pass. Thus I need to

mv GXPAM.XDS XPARM.XDS

and modify XDS.INP to read

JOBS= INTEGRATE CORRECT

Afterwards, another xds_par run gives the final intensities. Repeating this optimization sometimes helps.

High-remote

Due to a beamline problem, high-remote data collection stopped after 269 frames of 0.5° (the final frame is already affected). After restart of the beamline, another 100 frames were collected but they later turned out to merge badly with the first 269 frames - a hint that the monochromator was still heating up, or similar. So the latter frames were left out. The 269 frames are here (1.4 Gb).

From CORRECT.LP :

    a        b          ISa
6.595E+00  3.032E-04   22.36

...

      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

    8.21       20187    6913      7245       95.4%       2.9%      3.3%    20065   27.89     3.6%     2.9%    63%   1.625    3050
    5.84       36272   12417     12782       97.1%       5.5%      5.3%    36116   17.46     6.8%     7.0%    33%   1.196    5727
    4.78       46716   16015     16500       97.1%       6.8%      6.5%    46473   14.43     8.4%    10.0%    19%   1.004    7416
    4.15       55299   18949     19484       97.3%       7.5%      7.2%    55003   13.00     9.3%    11.7%    10%   0.896    8818
    3.71       63751   21798     22065       98.8%      12.2%     12.2%    63371    8.51    15.1%    20.2%     6%   0.819   10225
    3.39       70787   24180     24422       99.0%      19.5%     20.0%    70378    5.58    24.0%    33.3%     5%   0.786   11343
    3.14       61197   25100     26452       94.9%      32.5%     34.2%    57925    2.88    41.3%    63.9%     4%   0.740    9652
    2.94       40481   21869     28566       76.6%      53.5%     57.8%    33568    1.42    72.1%   112.8%    -2%   0.663    6208
    2.77       24584   16962     30228       56.1%      76.4%     82.4%    15055    0.77   107.5%   163.0%     2%   0.660    2828
   total      419274  164203    187744       87.5%      10.3%     10.4%   397954    8.06    12.9%    24.7%    14%   0.882   65267


NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  428770
NUMBER OF REJECTED MISFITS                            9102
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS                  0
NUMBER OF ACCEPTED OBSERVATIONS                     419668
NUMBER OF UNIQUE ACCEPTED REFLECTIONS               164343

Peak

360 frames (0.5° oscillation) at the peak wavelength were collected after the high-remote data. They can be downloaded from here (1.9 Gb). This peak dataset is somewhat difficult to index; if the results are really bad (e.g. distance refining far away from 370 mm) with the default 180 frames, then just try with 90 or 270 frames.

This is an excerpt from CORRECT.LP :

REFINED PARAMETERS:  DISTANCE BEAM ORIENTATION CELL AXIS                   
USING  166758 INDEXED SPOTS
STANDARD DEVIATION OF SPOT    POSITION (PIXELS)     1.80
STANDARD DEVIATION OF SPINDLE POSITION (DEGREES)    0.61
CRYSTAL MOSAICITY (DEGREES)     0.557
DIRECT BEAM COORDINATES (REC. ANGSTROEM)   0.001590 -0.003616  1.021443
DETECTOR COORDINATES (PIXELS) OF DIRECT BEAM    1536.60   1519.03
DETECTOR ORIGIN (PIXELS) AT                     1528.72   1536.95
CRYSTAL TO DETECTOR DISTANCE (mm)       370.85
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  0.999995 -0.001537 -0.002692
COORDINATES OF UNIT CELL A-AXIS   -41.622  -128.332    80.636
COORDINATES OF UNIT CELL B-AXIS    45.889    72.744   139.459
COORDINATES OF UNIT CELL C-AXIS  -551.193   220.474    66.370
REC. CELL PARAMETERS   0.006362  0.006103  0.001674  90.000  90.000  90.000
UNIT CELL PARAMETERS    157.174   163.848   597.351  90.000  90.000  90.000
E.S.D. OF CELL PARAMETERS  7.9E-01 8.7E-01 3.0E+00 0.0E+00 0.0E+00 0.0E+00
SPACE GROUP NUMBER     22

...

    a        b          ISa
7.764E+00  7.144E-04   13.43

...

      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

    8.29       26331    7014      7090       98.9%       5.3%      5.5%    26314   20.22     6.3%     4.2%    71%   1.936    3195
    5.90       48060   12542     12555       99.9%       8.4%      8.4%    48060   13.36     9.8%     8.5%    45%   1.322    5963
    4.83       61534   16074     16144       99.6%      11.0%     10.6%    61534   10.69    12.9%    13.2%    27%   1.051    7728
    4.19       71665   18994     19085       99.5%      12.5%     11.8%    71658    9.61    14.6%    16.0%    18%   0.920    9190
    3.75       77668   21598     21677       99.6%      19.5%     19.3%    77668    6.33    23.0%    27.1%     6%   0.794   10491
    3.42       78594   23767     23865       99.6%      28.3%     29.5%    78582    4.14    34.0%    45.0%     4%   0.735   11548
    3.17       64135   24351     26036       93.5%      42.7%     46.4%    60830    2.18    52.9%    78.5%     2%   0.689    9568
    2.97       40861   20207     27920       72.4%      63.8%     72.3%    35172    1.18    83.3%   118.6%     1%   0.657    6055
    2.80       23238   15074     29715       50.7%      89.5%    104.9%    15359    0.63   122.8%   175.5%     5%   0.646    2502
   total      492086  159621    184087       86.7%      14.8%     15.1%   475177    6.17    17.8%    30.6%    22%   0.901   66240


NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  501334
NUMBER OF REJECTED MISFITS                            8429
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS                  0
NUMBER OF ACCEPTED OBSERVATIONS                     492905
NUMBER OF UNIQUE ACCEPTED REFLECTIONS               159845

The verdict is clear: bad ISa, anomalous correlation > 30% only to about 5 A. The reason becomes clear if we load FRAME.cbf into XDS-Viewer, and zoom in: It is clear that these split reflections provide bad data. Fortunately it seems that in other areas of the detector, the reflections look better.

Inflection

360 frames (0.5° oscillation) at the inflection wavelength were collected after the peak data. They can be downloaded from here (1.8 Gb).