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).

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.

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).