As described in this chapter, rotation data images are processed in 8 steps
which are called in succession by XDS.
Information between the steps is communicated by files, which allows repetition of selected steps with a different set of input parameters without rerunning the whole program. The files generated by XDS are either ASCII type files that can be inspected and modified by using a text editor, or binary image files in the CBF format, a byte-offset variant of the CBFlib format. Such images are indicated by the file name extension ".cbf". All files have a fixed name defined by XDS, which makes it mandatory to process each data set in a newly created directory to avoid name clashes. Clearly, one should not run more than one XDS-job simultaneously in the same directory. Also, output files generated by rerunning selected steps (see Table 1) should first be given another name if their original contents are meant to be saved.
Data processing begins by copying an appropriate input file template into the data processing directory. Input file templates are provided with the XDS package for a number of frequently used data collection facilities. The copied input file must be renamed XDS.INP and edited to provide the correct parameter values for the actual data collection experiment.
All parameters in XDS.INP are named by keywords containing an equal sign as the last character, and many of them will be mentioned here in context to clarify their meaning. Execution of XDS invokes in succession each of the 8 program steps described below - or a subset of the steps named in the parameter JOB=. Results and diagnostics from each step are saved in files with the extension .LP attached to the program step name. These files should always be studied carefully to see whether processing was satisfactory or - in case of failure - to find out what could have gone wrong.
calculates lookup tables of spatial corrections for each detector pixel which are stored in the files X-CORRECTIONS.cbf and Y-CORRECTIONS.cbf . In subsequent data processing steps, when the true coordinates of a pixel with respect to the laboratory coordinate system are needed, the correction values for the X- and Y-coordinates are retrieved from the tables and added to the pixel's array coordinates in the data image.
Dependent on the detector, XYCORR computes the spatial corrections in different ways.
determines three lookup tables, saved as files BLANK.cbf, GAIN.cbf, and BKGINIT.cbf, that are required by the subsequent processing steps for classifying pixels in the data images as background or belonging to a diffraction spot ('strong' pixels).
Some detectors with insufficient protection from electromagnetic pulses may generate badly spoiled images whose inclusion leads to a completely wrong X-ray background table. These images can be identified in INIT.LP by their unexpected high mean pixel contents, and this step should be repeated with a different set of images.
locates strong diffraction spots occurring in a subset of the data images and saves their centroids on the file SPOT.XDS.
The data subset is defined by image number ranges where each range is specified by the keyword SPOT_RANGE=. COLSPOT identifies 'strong' pixels ( STRONG_PIXEL=) that are not in the background region ( BACKGROUND_PIXEL=). If the total number of 'strong' pixels occuring in the specified data images exceeds the upper limit as given by the input parameter MAXIMUM_NUMBER_OF_STRONG_PIXELS=, the weaker ones are discarded. Spots are defined as sets of 'strong' pixels adjacent in three dimensions. A spot is accepted if it contains a minimum number of 'strong' pixels ( MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT=) and if the spot centroid is sufficiently close to the location of the strongest pixel in the spot ( SPOT_MAXIMUM-CENTROID=).
Sharp edges like ice rings in the images can lead to an excessive number of 'strong' pixels erroneously classified as contributing to diffraction spots. These aliens could prevent IDXREF to recognize the crystal lattice.
uses the initial parameters describing the diffraction experiment as provided by XDS.INP and the observed centroids of the spots from the file SPOT.XDS to find the orientation, metric, and symmetry of the crystal lattice. IDXREF refines all or a specified subset of these parameters (input parameter REFINE(IDXREF)=). On return, the complete set of parameters are saved in the file XPARM.XDS, and the original file SPOT.XDS is replaced by a file of identical name - now with indices attached to each observed spot. Spots not belonging to the crystal lattice are given indices 0,0,0. XDS considers the run successful if the coordinates of at least 70% of the given spots can be explained with reasonable accuracy ( MAXIMUM_ERROR_OF_SPOT_POSITION=); otherwise XDS will stop with an error message. Alien spots often arise because of the presence of ice or small satellite crystals, and continuation of data processing may still be meaningful. In this case, XDS is called again with an explicit list of the subsequent steps specified in XDS.INP (input parameter JOB=).
To determine a crystal lattice that explains the observed locations of the diffraction spots listed in file SPOT.XDS, IDXREF proceeds as follows.
IDXREF uses the refined metric parameters of the reduced cell for testing each of the 44 possible lattice types (Kabsch, 1993). For each lattice type IDXREF reports the likelihood of being correct and the conventional cell parameters. IDXREF concludes with an overview of possible lattice symmetries (decision constants MAX_CELL_AXIS_ERROR=, MAX_CELL_ANGLE_ERROR=) but makes no automatic decision for the space-group. If the crystal symmetry is unknown, XDS will continue data processing with the crystal being described by its reduced cell basis vectors and triclinic symmetry.
recognizes regions in the initial background table (file BKGINIT.cbf) that are obscured by intruding hardware and marks the shaded pixels as untrusted. In addition, pixels outside a user-defined resolution range (INCLUDE_RESOLUTION_RANGE=) are marked and eliminated from the trusted region. The marked background table thus obtained is saved on file BKGPIX.cbf which is needed by the subsequent program steps.
For recognizing the obscured regions in the initial background, DEFPIX generates a control image (file ABS.cbf) that contains values around 10000 for unshaded pixels and lower values for shaded pixels. The classification of the pixels into reliable and untrusted ones is based on the two input parameters VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS= (default: 6000 30000) and INCLUDE_RESOLUTION_RANGE= (default: 20.0 0.0). Pixels in the table ABS.cbf with a value outside the ranges specified by the two parameters are marked unreliable (by -3) in the background table BKGPIX.cbf.
If the parameter VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS= specifies a too narrow value range, "good" regions will erroneously be excluded from the trusted detector region. Check BKGPIX.cbf with the XDS-Viewer program and, if necessary, repeat the DEFPIX step with more appropriate values.
supports the planning of data collection. It is based upon information provided by the input files XPARM.XDS, BKGPIX.cbf, X-CORRECTIONS.cbf and and Y-CORRECTIONS.cbf which become available on processing a few test images. XPLAN estimates the completeness of new reflection data, expected to be collected for each given starting angle ( STARTING_ANGLES_OF_SPINDLE_ROTATION=) and total crystal rotation ( TOTAL_SPINDLE_ROTATION_RANGES=), and reports the results for a number of selected resolution shells ( RESOLUTION_SHELLS=) in the file XPLAN.LP. To minimize recollection of data, the name of a file can be provided by the input parameter REFERENCE_DATA_SET=, the reference data set, which contains already measured reflections.
determines the intensity of each reflection predicted to occur in the rotation data images ( DATA_RANGE=) and saves the results on file INTEGRATE.HKL.
The diffraction parameters needed for predicting the reflection positions are initially provided by the file XPARM.XDS. These parameters are either kept constant or refined periodically using strong diffraction spots encountered in the data images. Whether refinement should be carried out at all and which parameters are to be refined can be specified by the user (input parameter REFINE(INTEGRATE)=). Centroids of the strong spots in the data images are computed from pixels that exceed the background by a given multiple of standard deviations (input parameters SIGNAL_PIXEL=, BACKGROUND_PIXEL=). Strong spots are used in the refinement if their centroids are reasonably close to their calculated position (input parameter MAXIMUM_ERROR_OF_SPOT_POSITION=).
For determination of the intensity, approximate values describing
extension and form of the diffraction spot must be specified. The
shapes of all spots become very similar when the contents of each of
their contributing image pixel is mapped into a 3-dimensional,
reflection-specific coordinate system centered on the surface of the
Ewald sphere, at the terminus of the diffracted beam wave vector (Kabsch, 1988b). In this coordinate system
alpha and beta span the plane tangential to the Ewald sphere with the
alpha-axis perpendicular to the incident- and the diffracted beam wave
vector. The gamma axis runs perpendicular to the alpha-axis and to the
rotated reciprocal lattice vector representing the reflection when the
Laue equations are satisfied. The number of grid points in this
coordinate system used for representing the transformed reflection
profile are usually chosen automatically by XDS; the user has the
option to override the automatic assignment by specifying the two input
The transformed spot can roughly be described as a Gaussian. Four parameters are used for this purpose:
All of the four parameters describing shape and extension of the spots can be determined automatically from the data images.
Integration is carried out by a two-step procedure. In the first pass,
spot templates are generated by superimposing profiles of fully
recorded, strong reflections, and all grid points with a value above a
minimum percentage of the maximum in the template ( CUT=) are defined as elements of
the integration domain. To allow for variations of their shape, profile
templates are generated from reflections located at nine regions of
equal size covering the detector surface and additional sets of nine to
cover equally-sized ( DELPHI=)
batches of images. The actual integration is carried out in the second
pass by profile fitting with respect to the spot shape determined in
the first pass. Reflections with less than
of observed reflection intensity will be discarded. Otherwise, the
missing intensity is estimated from the learned reflection profiles.
On return from the INTEGRATE step, the data image last processed with all expected spots encircled is saved in the file FRAME.cbf for inspection with help of the XDS-Viewer program.
applies correction factors to intensities and standard deviations of all reflections found in the file INTEGRATE.HKL, determines the space group if unknown and refines the unit cell constants, reports the quality and completeness of the data set, and saves the final integrated intensities on the file XDS_ASCII.HKL.
CORRECT accepts reflections from file INTEGRATE.HKL that are
Thus, the user has the option to exclude unreliable reflections from
the final data set by repeating the CORRECT step with appropriate
Intensities of the accepted reflections are first corrected for effects due to polarization of the incident beam (parameters FRACTION_OF_POLARIZATION=, POLARIZATION_PLANE_NORMAL=) and absorption effects (parameter AIR=, SILICON=, SENSOR_THICKNESS=) arising from differences in path lengths of the diffracted beam. These corrections do not depend on knowledge of the space group.
The integrated intensities of the reflections on file INTEGRATE.HKL may or may not have been indexed in the correct space group; for the purpose of integration it is important only that all reflections occurring in the data images have been located exactly and indexed using some unit cell basis. The correct reflection indices in the true space group are always a linear transformation of the original indices used in INTEGRATE.HKL. All lattices consistent with the locations of the reflections saved in INTEGRATE.HKL (decision parameters MAX_CELL_AXIS_ERROR=, MAX_CELL_ANGLE_ERROR=) and their corresponding linear transformations are printed to provide a useful overview similar to the one shown in IDXREF.LP.
If the space group is not specified, XDS proposes one of the enantiomorphous space groups without screw axes that is compatible with the observed lattice symmetry and explains the intensities of a subset of the reflections (TEST_RESOLUTION_RANGE=) at an acceptable Rmeas (Diederichs and Karplus, 1997) using a minimum number of unique reflections. The criteria for an acceptable Rmeas are controlled by the decision parameters MIN_RFL_Rmeas=, and MAX_FAC_Rmeas=.
The user can always override the automatic decisions by specifying the
correct space group number (SPACE_GROUP_NUMBER=)
and unit cell constants (UNIT_CELL_CONSTANTS=)
in XDS.INP and repeating the CORRECT step. This provides a simple way to
rename orthorhombic cell constants if screw axes are present.
In addition, the user has the option to specify in XDS.INP
For refinement of the unit cell constants (parameter REFINE(CORRECT)=), CORRECT uses a subset of the accepted reflections, whose observed centroid is sufficiently close to the predicted spot position (parameter MAXIMUM_ERROR_OF_SPOT_POSITION=). The refined set of parameters is saved on file GXPARM.XDS which has the identical layout as file XPARM.XDS produced by IDXREF. If the crystal has not slipped during data collection, these parameters are quite accurate.
Other correction factors which partially compensate for radiation damage, absorption effects, and variations in sensitivity of the detector surface are determined from symmetry-equivalent reflections usually found in the data images. The corrections are chosen such that the integrated intensities of symmetry-equivalent reflections come out as similar as possible. The user may control application of the various corrections by specifying the parameter CORRECTIONS= by a combination of the keywords DECAY MODULATION ABSORPTION. Whether Friedel-pairs are considered as symmetry-equivalent reflections in the calculation of the correction factors depends on the values of the two parameters STRICT_ABSORPTION_CORRECTION= and FRIEDEL'S_LAW=. The number of correction factors is controlled by the input parameters MINIMUM_I/SIGMA=, NBATCH=, and REFLECTIONS/CORRECTION_FACTOR=.
The residual scatter in intensity of symmetry-equivalent reflections is used to estimate their standard deviations. Here, the initial estimate v0(I) (obtained from the INTEGRATE step) for the variance of the reflection intensity I is replaced by v(I)=a*(v0(I)+b*I^2). The two constants a and b are chosen to minimize discrepancies between v(I) and the variance estimated from sample statistics of symmetry related reflections. Based on the more realistic error estimates for the intensities, outliers are recognized by comparison with other symmetry-equivalent reflections. These outliers are included in the main output file XDS_ASCII.HKL in which they are marked by a negative sign attached to the estimated standard deviations of their intensity. Classification of a reflection as a misfit is controlled by a decision constant which has the default value of WFAC1=1.5. A lower value like WFAC1=1.0 specified by the user will lead to an increasing number of misfits and lower R-factors as outliers are not included in the reported statistics.
Data quality as a function of resolution is described by the agreement of intensities of symmetry-related reflections and quantified by the R-factors, Rsym, and the more robust indicator, Rmeas (Diederichs and Karplus, 1997). These R-factors as well as the intensities of all reflections with indices of type h 0 0, 0 k 0, and 0 0 l and those expected to be systematically absent provide important information for identification of the correct space-group. Clearly, large R-factors or many rejected reflections (MISFITS) or large observed intensities for reflections expected to be systematically absent suggest that the assumed space-group or the indexing is incorrect. The presence or absence of anomalous scatterers is specified by the parameter FRIEDEL'S_LAW=.
Finally, CORRECT analyzes the distribution of reflection intensities as a function of their resolution and reports outliers from the Wilson plot. Often these aliens arise from ice rings in the data images. To suppress the unwanted reflections from the final output file XDS_ASCII.HKL, the user copies them to a file named REMOVE.HKL in the current directory and repeats the CORRECT step.
© 2009-2013, MPI for Medical Research, Heidelberg Imprint. Wolfgang.Kabsch@mpimf-heidelberg.mpg.de