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This article deals with how to process serial synchrotron crystallography (SSX) data. | |||
The particular data we are processing are artificial and were prepared by James Holton. The files Illuin_microfocus_minimal_00[1-3].tar.bz2 can be [http://bl831.als.lbl.gov/example_data_sets/tarballs downloaded] and the data and problem are described on his [http://bl831.als.lbl.gov/~jamesh/challenge/microfocus microfocus challenge page], and in a [http://journals.iucr.org/d/issues/2019/02/00/ba5297/index.html paper]. | |||
The challenges are | |||
# partial data sets: each of the 100 data sets has only 3 good frames of 1° oscillation; later frames have strong radiation damage | |||
# the crystals decay to about 1/2 within these 3 frames | |||
# the b and c axes are the same length, but the simulated crystals are orthorhombic. This makes it difficult to index them consistently - it is wrong to just merge them in a orthorhombic space group without resolving the indexing ambiguity, because that yields a pseudo-tetragonal twinned merged data set. | |||
== Round 1: processing the data, and determining the space group == | == Round 1: processing the data, and determining the space group == | ||
In order to be able to merge the data in XSCALE, we must ensure that they are all processed in the same space group, with similar cell parameters. Some exploratory processing (not shown) and averaging of cell parameters reveals that IDXREF finds a primitive lattice with one axis of 38.3 Å, and two with 79.1 Å; angles are 90°. The data go to 1.8 Å; beyond that, the intensities suddenly drop to 0 - presumably because James Holton simulated them only that far. | |||
Using the following as the processing script integrate.rc: | Using the following as the processing script integrate.rc: | ||
<pre> | <pre> | ||
| Line 28: | Line 38: | ||
FRIEDEL'S_LAW=TRUE | FRIEDEL'S_LAW=TRUE | ||
eof | eof | ||
find $PWD/../wedge* -name XDS_ASCII.HKL | awk '{print "INPUT_FILE=",$0;print "NBATCH=1 CORRECTIONS= | find $PWD/../wedge* -name XDS_ASCII.HKL | awk '{print "INPUT_FILE=",$0;print "NBATCH=1 CORRECTIONS=ALL"}' >> XSCALE.INP | ||
</pre> | </pre> | ||
we obtain in P1 | we obtain in P1 | ||
| Line 116: | Line 126: | ||
REIDX=0 1 0 0 0 0 1 0 1 0 0 0 | REIDX=0 1 0 0 0 0 1 0 1 0 0 0 | ||
</pre> | </pre> | ||
where the last line takes care of the shuffling of axes into the order k,l,h, , and obtain | where the last line takes care of the shuffling of axes into the order k,l,h, (after all, the XDS_ASCII.HKL are in P1 with a,b,c of 38.3,79.1,79.1) , and obtain | ||
<pre> | <pre> | ||
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION | SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION | ||
| Line 146: | Line 156: | ||
Analysis with | Analysis with | ||
xscale_isocluster -dim 2 -clu 2 temp.ahkl | xscale_isocluster -dim 2 -clu 2 temp.ahkl | ||
yields a iso.pdb which is not at all a single cluster; it is a severely elongated single cloud. We must now investigate whether the data have lower than tetragonal symmetry. | yields a iso.pdb which is not at all a single cluster; it is a severely elongated single cloud: | ||
[[File:1g1c-94.png]] | |||
(If the space group were correct, the result of [[xscale_isocluster]] should look similar to this: | |||
[[File:Lyso-xscale-isocluster.png]] | |||
which is from a lysozyme SSX data collection performed at the SLS; outliers are labelled. In this case, the data are truely tetragonal.) | |||
We must now investigate whether the data have lower than tetragonal symmetry. | |||
XSCALEing with | XSCALEing with | ||
SPACE_GROUP_NUMBER=16 | SPACE_GROUP_NUMBER=16 | ||
| Line 158: | Line 178: | ||
cluster: 2 center: 6 elements: 49 core: 49 halo: 0 | cluster: 2 center: 6 elements: 49 core: 49 halo: 0 | ||
</pre> | </pre> | ||
and prepares XSCALE.1.INP (and XSCALE.2.INP for further use. | and prepares XSCALE.1.INP (and XSCALE.2.INP) for further use (these two files collect the differently, but internally-consistently indexed XDS_ASCII.HKL files). | ||
coot iso.pdb | coot iso.pdb | ||
shows | shows | ||
thus two well separated clouds. | [[File:Coot.png]] | ||
and thus reveals two well separated clouds, corresponding to the two possible indexing modes of the data in an orthorhombic space group. | |||
Using XSCALE.1.INP with its 51 XDS_ASCII.HKL, and | Using XSCALE.1.INP with its 51 XDS_ASCII.HKL, and FRIEDEL'S_LAW=TRUE, we get | ||
<pre> | <pre> | ||
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION | SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION | ||
| Line 195: | Line 217: | ||
xdscc12 -w XSCALE.1.HKL | grep ^a | sort -nk6 | xdscc12 -w XSCALE.1.HKL | grep ^a | sort -nk6 | ||
and find that data sets 1 and 17 are wrongly included in the cloud of 51 data sets. Thus they are removed manually from XSCALE.INP. | and find that data sets 1 and 17 are wrongly included in the cloud of 51 data sets. Thus they are removed manually from XSCALE.INP. | ||
We then re-run XSCALE with MERGE=TRUE. The resulting XSCALE.1.HKL is then used as REFERENCE_DATA_SET for a second round of integration with XDS. | |||
After <code>xscale_isocluster -dim 2 -clu 1</code> , | |||
coot iso.pdb | |||
now reveals a single cloud: | |||
[[File:1g1c-19.png]] | |||
We then re-run XSCALE with MERGE=TRUE. The resulting reflection output file XSCALE.1.HKL is then used as REFERENCE_DATA_SET for a second round of integration with XDS. | |||
pointless xdsin XSCALE.1.HKL | pointless xdsin XSCALE.1.HKL | ||
| Line 238: | Line 267: | ||
thus we now know the spacegroup. | thus we now know the spacegroup. | ||
== Round 2: using the REFERENCE_DATA_SET == | == Round 2: using the REFERENCE_DATA_SET obtained from one cluster== | ||
The processing script integrate.rc is changed a bit: | The processing script integrate.rc is changed a bit, to a) use the REFERENCE_DATA_SET, b) prevent adjustment of variances by CORRECT (this should rather be done by XSCALE) , c) allow some radiation damage correction in XSCALE: | ||
<pre> | <pre> | ||
#!/bin/bash -f | #!/bin/bash -f | ||
| Line 301: | Line 330: | ||
total 256288 42935 43227 99.3% 13.4% 14.0% 255575 11.63 14.6% 99.6* 21* 0.975 19321 | total 256288 42935 43227 99.3% 13.4% 14.0% 255575 11.63 14.6% 99.6* 21* 0.975 19321 | ||
</pre> | </pre> | ||
The structure can now easily be solved with hkl2map | The substructure (locating 4 Se with anom data to 3Å) and structure (198 residues) can now easily be solved with [[ccp4com:SHELX C/D/E|hkl2map]]: | ||
== Result == | |||
=== SHELXC: anomalous CC<sub>1/2</sub> === | |||
[[File:Cc12ano.png]] | |||
=== SHELXD: CCall ''versus'' CCweak, and histogram === | |||
[[File:Ccallcsccweak.png]] | |||
[[File:Histcfom.png]] | |||
=== SHELXE: contrast versus cycle, and PDB with structure === | |||
[[File:Contrastvscycle.png]] | |||
[[File:Ribbon.png]] | |||
Further optimization of processing may be possible, but is left as an exercise to the reader. | |||