1、Practical Approaches to Data Processing Using XDSKay DiederichsProtein Crystallography / Molecular BioinformaticsOverviewXDS is a data reduction program for X-ray data collected by the oscillation method on area detectors Author: Wolfgang Kabsch (MPI Heidelberg)Information flow within XDSUsage; Opti

2、mization; InterfacesXDSwiki; referencesDemonstration: processing of dataset (e.g. Wladek Minors corresponding to PDB 1WQ6)Summary and questionsthroughout this talk: program, file2The XDS program suitesource code available from :XDS-Viewer : inspect control images written by XDS, or (single) data fra

3、mes (alternatively, latest adxv may be used)binary distribution (by W. Kabsch) for Linux & Mac from :/ mpimf-heidelberg.mpg.de/kabsch/xds/:XDS: the main program (indexing, integrating, scaling)XSCALE: scale several XDS intensity data sets together, statisticsXDSCONV: convert to CCP4, CNS, SHELX, . f

4、ormatmy own programs: XDSSTAT, generate_adx (both in XDSwiki)3AlgorithmsUnique features:3D - profiles of reflections transformed into their own coordinate systems makes them highly similarPixel-labelling methodSmooth scalingRobust estimation of parameters throughoutRadiation-damage correction (XSCAL

5、E)4How to use XDS ?Prepare a single input file with parameters describing data reduction often written by beamline softwareParameters and their keywords have the form e.g. DETECTOR_DISTANCE= 120.There are about 30 relevant parameters, but only about 15 are required (and change between projects). All

6、 parameters have reasonable defaults where possible.Quick start: from XDSwiki5 JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT ORGX=1546 ORGY=1552 !Detector origin (pixels); e.g. NX/2 NY/2 DETECTOR_DISTANCE=180 !(mm) !degrees (0) !Angstroem NAME_TEMPLATE_OF_DATA_FRAMES=frms/wga2-27_1_?.img

7、DATA_RANGE=1 360 !Numbers of first and last data image collected BACKGROUND_RANGE=1 10 !Numbers of first and last data image for background SPACE_GROUP_NUMBER= 19 !0 for unknown crystals; cell constants are ignored. UNIT_CELL_CONSTANTS= 44.4 86.4 104.5 90 90 90 REFINE(IDXREF)=BEAM AXIS ORIENTATION C

8、ELL DISTANCE REFINE(INTEGRATE)=DISTANCE BEAM ORIENTATION CELL ! AXIS FRACTION_OF_POLARIZATION=0.99 ! SLS X06SA DETECTOR=CCDCHESS MINIMUM_VALID_PIXEL_VALUE=1 OVERLOAD=65000 VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS= 7000 30000 !Used by DEFPIX !for excluding shaded parts of the detector. INCLUDE_RESOLUT

9、ION_RANGE=50.0 1.3 !Angstroem; used by DEFPIX,INTEGRATE,CORRECTExample for MarCCD 225 SLS PX-IIIBold keyword/parameter pairs are required. Complete documentation at Templates for many detectors at6Using XDS - principles Isimple, if basic idea is understoodThere is one JOB= line in which does not spe

10、cify a parameter, but instead a list of tasks:JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT data reduction is divided into tasks/jobs in modular way information storage/exchange/flow between tasks by data files which may be inspected/analyzed each task needs the result from the previous t

11、asks fine-tuning of a task does not require previous tasks to be repeated each task write its output file .LP7Using XDS - principles IIXYCORR : write positional correction files ( , Y-CORRECTIONS.cbf )INIT : find background pixels (defaults usually OK)COLSPOT: find reflection positionsIDXREF : index

12、 reflections; user may supply/choose spacegroupXPLAN not required : strategy for data collectionDEFPIX : find beamstop shadow (defaults mostly OK)INTEGRATE : evaluates intensities on all frames, writes and CORRECT : scales, rejects outliers, statistics, writes XDS_ASCII.HKL (and other files)8Informa

13、tion flowXYCORRINITCOLSPOTIDXREFDEFPIXINTEGRATECORRECTXPARM.XDSGXPARM.XDS GX-CORRECTIONS.cbfGY-CORRECTIONS.cbf BKGINIT.cbfSPOT.XDSBKGPIX.cbfABS.cbfINTEGRATE.HKLFRAME.cbfXSCALEXDSCONVORGXORGYDETECTOR_DISTANCEX_RAY_WAVELENGTHSPACE_GROUP_NUMBERDATA_RANGENAME_TEMPLATE_OF_DATA_FRAMESOSCILLATION_RANGEXDSS

14、TATpointlessloggraph9XDS output file: !OUTPUT_FILE=INTEGRATE.HKL DATE= 3-Oct-2006!Generated by INTEGRATE (XDS VERSION August 18, 2006) !PROFILE_FITTING= TRUE !SPACE_GROUP_NUMBER= 92!NAME_TEMPLATE_OF_DATA_FRAMES= ./series_2_?.img !DETECTOR=ADSC !STARTING_FRAME= 1!NUMBER_OF_ITEMS_IN_EACH_DATA_RECORD=2

15、0!H,K,L,IOBS,SIGMA,XCAL,YCAL,ZCAL,RLP,PEAK,CORR,MAXC,! XOBS,YOBS,ZOBS,ALF0,BET0,ALF1,BET1,PSI!Items are separated by a blank and can be read in free-format!END_OF_HEADER -45 -9 -60 -3.755E+01 4.144E+01 273.5 0.75268 100 -10 46 -45 -9 -59 8.133E+00 4.372E+01 274.5 0.75525 100 10 46 0.0 -49.52 0.16 45

16、.34 49.22 -29.95 -45 -8 -60 6.502E+01 4.327E+01 271.3 0.75438 100 14 47 272.0 -49.52 0.16 45.26 49.23 -30.66 10XDS output file: !FORMAT=XDS_ASCII MERGE=FALSE FRIEDELS_LAW=TRUE !OUTPUT_FILE=XDS_ASCII.HKL DATE= 3-Oct-2006!Generated by CORRECT (XDS VERSION August 18, 2006) !PROFILE_FITTING= TRUE !SPACE

17、_GROUP_NUMBER= 92!NAME_TEMPLATE_OF_DATA_FRAMES= ./series_2_?.img !DATA_RANGE= 1 399!STARTING_FRAME= 1!DETECTOR=ADSC !NUMBER_OF_ITEMS_IN_EACH_DATA_RECORD=12!ITEM_H=1!ITEM_K=2!ITEM_L=3!ITEM_IOBS=4!ITEM_SIGMA(IOBS)=5!ITEM_XD=6!ITEM_YD=7!ITEM_ZD=8!ITEM_RLP=9!ITEM_PEAK=10!ITEM_CORR=11!ITEM_PSI=12!END_OF_

18、HEADER 0 0 4 0 0 -4 0 0 5 11XDS : feedback of information from later steps to previous steps (postrefinement)To optimize data quality, you may try to rename (written by CORRECT) to copy 2 lines of INTEGRATE output: from to run the DEFPIX/INTEGRATE/CORRECT steps again this improves statistics quite a

19、 bit if geometry not accurately known on 1st pass.More in XDSwiki (article Optimization“)12Visualizing Distortions and scaling problemsXDS writes .cbf files for control purposesXDS-Viewer (or adxv) can display these filesIf not corrected: systematic errors, many rejections, reduced data quality, bad

20、 anomalous signal13X/Y- distortions (from CORRECT task) has 10*(xobs-xcal) as a function of positionSimilar for y: 14Further information from XDSSTATwrites XDSSTAT.LP (visualize with CCP4 loggraph) shows scale factor in percent as a function of position (after correction in XDS) shows outliers mappe

21、d on detector shows R-factor mapped on detector shows anomalous difference mapped on detector These files may be displayed with adxv, XDS-Viewer, or VIEW (distributed with old versions of XDS)15 Frame #refs #misfits Iobs sigma Iobs/sigma Peak Corr Rmeas #rmeas #unique 1 11434 96 137. 21.0 6.53 97.97

22、 42.97 0.1419 11429 5 2 8727 107 125. 19.9 6.27 99.86 41.05 0.1434 8725 2 3 8826 58 131. 20.6 6.36 99.86 41.05 0.1353 8824 2 4 8636 116 127. 20.1 6.31 99.89 40.57 0.1361 8633 3 5 8776 59 131. 20.8 6.30 99.06 40.06 0.1287 8773 3 6 8713 78 132. 21.1 6.24 99.61 38.41 0.1426 8710 3. R_d factor as a func

23、tion of frame number difference framediff n-all Rd-all n-notfriedel Rd-notfriedel n-friedel Rd-friedel 0 26160 0.1720 10856 0.1698 15304 0.1736 DIFFERENCE 1 51943 0.1738 21047 0.1695 30896 0.1768 DIFFERENCE 2 50238 0.1626 20888 0.1648 29350 0.1612 DIFFERENCE 3 47429 0.1645 20297 0.1639 27132 0.1649

24、DIFFERENCE 4 46395 0.1679 20095 0.1695 26300 0.1666 DIFFERENCE 5 44861 0.1649 19505 0.1665 25356 0.1637 DIFFERENCE 6 43656 0.1633 19279 0.1658 24377 0.1615 DIFFERENCE.16InterfacesGUIs: XDSi (P. Kursula; M. Krug)CCP4: pointless, (combat), xdsconv (type CCP4 or CCP4_I): xdsconvpipelines: xia2 (CCP4),

25、autoPROC (Globalphasing), autoxds (SSRL), .17XDS ReferencesKabsch, W. (1988). Evaluation of single-crystal X-ray diffraction data from a position-sensitive detector. J. Appl. Cryst. 21, 916-924. Kabsch, W. (1993). Automatic processing of rotation diffraction data from crystals of initially unknown s

26、ymmetry and cell constants. J. Appl. Cryst. 26, 795-800. Kabsch, W. (2001) Chapter 11.3. Integration, scaling, space-group assignment and post refinementKabsch, W. (2001) Chapter 25.2.9. XDSboth in International Tables for Crystallography, Volume F. Crystallography of Biological Macromolecules, Ross

27、mann, M.G. and Arnold, E. (2001). Editors. Dordrecht: Kluwer Academic Publishers.Kabsch, W. (2010). XDS. Acta Cryst. D66, 125-132. (open access)Kabsch, W. (2010). Integration, scaling, space-group assignment and post-refinement. Acta Cryst. D66, 133-144. (open access)18XDSwikistarted Feb 2008; 100 p

28、ages ate.g. Optimization“; explanations of task outputTips and Tricks“Quality Control“ with datasets and resultsanybody can contribute (same holds for CCP4wiki: 220 pages at)19Know what tools are available!Robust processing even if mosaicity highfast: parallel processing possible (synchrotron !)can

29、run on ASCII terminal, over a slow line (but needs X11 terminal if difficulties arise)transparent decompression of framesNot all parts of the frame header are read: distance, wavelength, beam position, -phi must be supplied by the user (or the beamline software)no/little visualization (compared to M

30、OSFLM, d*Trek and HKL)20Some typical questions .“How to scale & merge different datasets from similar or same xtal(s), using XDS?”“What about twinning? Is it possible to integrate small molecule data as well?”“Does XDS correct for radiation damage (increased B factors) without scaling all to the first data set?” “Will an easier to use masking system be developed?”More Qs and As