Merging
The result of Indexing and integration is a set of Python pickle files, each of which essentially contains a table of Miller indices of the observed reflections, their integrated intensities, and estimated errors. In the general case, these files reflect the measurements from single shots, each exposing different crystals with a unique pulse of X-rays. Merging refers to the procedure applied to unite all these observations into a single data set. During merging, a distinct multiplicative factor, which accounts for the variance in pulse intensity and crystal size, is applied to the observations from a single shot to bring all the observations onto a common scale. After rejecting possible outliers, the intensities for individual reflections are summed, and their errors are propagated in quadrature. The result of merging is an mtz file suited for further processing, e.g. molecular replacement.
Merging a set of integration files
In cctbx.xfel the per-image scale factors are determined using a scaling reference. This scaling reference is expected to be a previously solved, isomorphous data set. The scale factor is determined by a least-squares fit of the observations to the reference intensities, after applying corrections for polarization<ref>Kahn, R, et al. Macromolecular Crystallography with Synchrotron Radiation: Photographic Data Collection and Polarization Correction. J Appl Cryst 15, 330–337 (1982).</ref>, and a significance filter, which limits the resolution of each diffraction pattern based on the signal-to-noise ratio. Lattices not conforming to the Bravais symmetry of the scaling reference are rejected. By default non-isomorphous lattices with cell lengths differing by more than 10% in length from the mean, or 2° in the angles are also rejected, as are lattices that correlate poorly with the scaling reference.
cxi.merge may take several passes over the integrated images. In order to speed up processing, cxi.merge will write the scaled data to a back end database during each pass. Currently three database back ends are implemented.
FSis the simplest back end. It stores the scaled intensities, their Miller indices, and information about the shots they were observed during in three flat files on the file system.
- The
MySQLback end stores data in a MySQL database. The database must be set up beforehand, and credentials to access it must be supplied in the parameters passed to cxi.merge.
- The
SQLiteback end uses a simple SQLite database, which is written to a single file on the file system. It is easier to use than theMySQLback end and more efficient than theFSbackend. Regrettably, theSQLiteback end does not appear to work on the Lustre file system.
Compared to indexing and integration, merging is a relatively quick procedure. However, particularly for large datasets, it may significantly strain computational resources. Therefore, it is recommended to merge data on SLAC's interactive nodes.
$ ssh psanacs.slac.stanford.edu $ cd myrelease
In cctbx.xfel images are merged using the cxi.merge command. The results are logged to standard output, so it is advisable to redirect the stream to a file.
$ cxi.merge Ls04-lysozyme-merge.phil > merge.out
Here, Ls04-lysozyme-merge.phil is a phil-file with the parameters to control the merging procedure. In this tutorial only a subset of the available options are defined.
- backend
- Back end database;
FSfor flat-file ASCII data storage,MySQLandSQLitefor the respective proper database back ends. - d_min
- Limiting resolution for scaling and merging
- data
- Directory containing integrated data in pickle format. Repeat to specify additional directories.
- merge_anomalous
Trueto merge anomalous contributors (i.e. Bijvoet mates),Falseto preserve them- min_corr
- Correlation cutoff for rejecting individual frames
- model
- The scaling reference, PDB filename containing atomic coordinates and isomorphous
CRYST1record - nproc
- Specifies the number of scaling processes cxi.merge may have running at any one time
- output.prefix
- Prefix for all output file names
- rawdata.sdfac_auto
Trueto applySDFACcorrection to each image, assuming negative intensities are normally distributed noise- rescale_with_average_cell
- Rescale the images a second time, requiring images to conform to the average unit cell. If set to
True,set_average_unit_cellmust also be set toTrue. - set_average_unit_cell
- If
Trueset the unit cell of the merged data to the average of the merged images, otherwise use the unit cell of the scaling reference
The merged data is written to an mtz-file named output.prefix.mtz. Standard output, redirected to merge.out above, mainly consists of statistics for each individual image as it is scaled. The output concludes with a section labelled FINISHED MERGING, which first lists the number of accepted and rejected images,
962 of 1185 integration files were accepted 127 rejected due to wrong Bravais group 1 rejected for unit cell outliers 12 rejected for low signal 83 rejected due to poor correlation
This is followed by a histograms of the unit cell distribution, and the merging table:
---------------------------------------------------------------------------------
<asu <obs
Bin Resolution Range Completeness redun> redun> n_meas <I> <I/sig(I)>
---------------------------------------------------------------------------------
1 -1.0000 - 6.4633 [306/309] 67.61 68.28 20893 35621 27.044
2 6.4633 - 5.1299 [285/285] 43.18 43.18 12305 19272 12.490
3 5.1299 - 4.4814 [275/275] 46.77 46.77 12862 24391 14.261
4 4.4814 - 4.0716 [269/269] 43.38 43.38 11670 29436 14.819
5 4.0716 - 3.7798 [249/249] 35.50 35.50 8840 29811 12.716
6 3.7798 - 3.5569 [267/267] 32.07 32.07 8562 24212 10.523
7 3.5569 - 3.3787 [274/274] 20.99 20.99 5750 23626 8.171
8 3.3787 - 3.2317 [256/256] 15.96 15.96 4085 24168 6.746
9 3.2317 - 3.1072 [263/264] 12.16 12.20 3209 23936 5.859
10 3.1072 - 3.0000 [263/263] 8.74 8.74 2298 20613 4.556
All [2707/2711] 33.37 33.42 90474 25594 11.978
----------------------------------------------------------------------------------
In this case, the overall completeness to 3.0 Å is 2,707 / 2,711, or approximately 100%, and each observed reflection is measured 33.42 times on average (<obs redun>). In total 90,474 observations, with an average scaled and integrated intensity of 25,594 analog-to-digital units (ADU), were merged and the mean I / σ(I) was determined to be 11.978.
Additional merging statistics
cxi.xmerge retrieves the scaled, unmerged intensities from the database back end, and calculates the CC1/2<ref>Karplus, P. A. & Diederichs, K. Linking Crystallographic Model and Data Quality. Science 336, 1030–1033 (2012).</ref> and CCiso statistics. CC1/2 is defined as Pearson's correlation coefficient between two sets, such that for each unique reflection the average intensities of two randomly chosen halves of its independent observations are assigned to different set. CCiso is the correlation coefficient between the merged data and the isomorphous scaling reference. Both statistics are computed in each resolution bin, as well as for the full set of reflections.
Once the database has been populated using cxi.merge, cxi.xmerge can be run, using the parameters defined in a phil-file, Ls-04-lysozyme-xmerge.phil.
$ cxi.xmerge Ls04-lysozyme-xmerge.phil
The options used in this tutorial not already described in Merging a set of integration files are explained below.
- scaling.mtz_column_F
- Column name in the reference structure mtz-file with structure factors
- scaling.mtz_file
- mtz-file with reference structure factors, must have data type
F - scaling.log_cutoff
- Intensities less than e
scaling.log_cutoffwill not be included in the calculation
The results are printed on standard output and conclude with a table including the CC
---------------------------------------------------------------------------------------
CC CC R R Scale Scale
Bin Resolution Range Completeness int N iso N int iso int iso
---------------------------------------------------------------------------------------
1 -1.0000 - 6.4633 [233/309] 45.1% 233 56.3% 142 57.5% 45.6% 0.749 3.084
2 6.4633 - 5.1299 [184/285] 22.5% 184 56.7% 62 57.2% 31.5% 0.862 2.841
3 5.1299 - 4.4814 [203/275] 43.7% 203 57.6% 85 55.3% 37.0% 0.830 2.977
4 4.4814 - 4.0716 [220/269] 39.0% 220 59.7% 101 51.4% 30.3% 0.805 3.296
5 4.0716 - 3.7798 [187/249] 77.5% 187 33.6% 63 54.3% 39.4% 0.775 4.243
6 3.7798 - 3.5569 [198/267] 35.4% 198 47.2% 56 56.8% 27.5% 0.903 3.587
7 3.5569 - 3.3787 [198/274] 32.0% 198 70.7% 40 59.9% 31.4% 0.814 3.846
8 3.3787 - 3.2317 [163/256] 30.6% 163 24.5% 28 63.9% 49.1% 0.734 4.278
9 3.2317 - 3.1072 [164/264] 40.6% 164 53.6% 7 80.4% 32.1% 0.622 5.543
10 3.1072 - 3.0000 [137/261] 8.2% 137 93.9% 5 85.6% 33.4% 0.221 4.372
All 34.7% 1887 47.7% 589 62.5% 39.5% 0.754 3.157
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In this case the overall CC1/2 (CC int) is 34.7%, and the correlation coefficient to the isomorphous reference, CCiso (CC iso) is 47.7%.
References
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