LAUE SUITE OVERVIEW - PROCESSING STAGES ======================================= INTRODUCTION This document gives an overview of the Laue data processing software suite developed at the Daresbury Laboratory. A general overview of the development of the suite is given in reference 1. An account of the principles used, in the majority of the programs in the Laue software suite, is given in Helliwell et. al (reference 6). Further papers relating to the development of the Laue data processing software are given in references 2 to 5, 9 and 10. It should be noted that some of the basic framework is derived from the Cambridge/Imperial College suite of programs for the processing of monochromatic oscillation photographs of protein crystals (refs. 7, 8). Many of the original programs are no longer normally needed as they have been superseded by more recently developed programs in particular LAUEGEN (refs. 3, 9) and LSCALE (ref. 10). List of sections: Overview of The Stages for Intensity Measurements Processing of Difference Data Summary of Main Programs and Files Involved Prediction of the Laue Patterns Determination of the Crystal Orientation Parameter Refinement Intensity Measurement Scaling Within (Film) Packs Wavelength Normalisation Absorption Correction Unscrambling Harmonics Programs for Processing Difference Data References OVERVIEW OF THE STAGES FOR INTENSITY MEASUREMENTS This section outlines the stages involved in the processing of Laue data. The sequence of the processing stages is also illustrated in the accompanying flowchart. Figure 1 Flowchart of Main Processing Sequence (at end of chapter) The stages in processing the data are as follows: * Prediction of Laue patterns. This is useful for studying the nature of Laue patterns and may be helpful in determining the crystal orientation. * Determining the crystal orientation. This may be done manually but more often it is achieved using an auto-indexing procedure based on the identification of nodal spots. * Refinement of the cell and setting parameters for the crystal and making an accurate prediction of the spot positions on the images. * Integration of the optical densities at the predicted spot positions for all the images. * Scaling the Laue data. This includes pack and plate scaling, wavelength normalistion, absorption correction and merging of reflection data. In addition other intensity corrections such as the Lorentz and polarisation corrections are applied. The wavelength normalisation is usually done by using an internal scaling procedure based on symmetry equivalent reflections measured at different wavelengths. An alternative method involves scaling against a reference set of data. * Deconvolution of reflection intensities which are harmonic overlaps (multiples). This is based on using symmetry equivalent reflections recorded at different wavelengths * The merging of intensity data may be done using the appropriate Laue processing program or may be done externally to the suite using programs from the CCP4 program suite. PROCESSING OF DIFFERENCE DATA When proccessing difference data, it would of course be possible to process each set of Laue data to a merged set of intensities (or F's) as described in the previous section and then scale the data sets together using one of the programs normally used in monochromatic work. However, an alternative strategy is available when Laue images can be obtained for the two crystal states with the crystal in the same orientation. Fractional differences are calculated and each single reflection is individually scaled to the corresponding reflection in a reference set of data corresponding to the initial state of the crystal. This may be a monochromatic data set or a set of Laue data processed as described in the section above. The procedure is usually carried out by following stages up to the intensity integration as described in the previous section followed by use of the program DIFFLAUE . SUMMARY OF MAIN PROGRAMS AND FILES INVOLVED This summary shows the programs and files involved in Laue data processing using the most recently developed programs in the Laue software suite. Start | [.ldm file] | - may be created by LAUEGEN .img files | | LAUEGEN | | | .mtz file | (or .ge1/.ge2) | | | --------------------------------------- | | Difference .ge1 or .mtz | | Data files | .ge1 file | .lsp file | [another .ge1] | | [.ctl file] | | [.mtz file] | | | LSCALE DIFFLAUE | | | | | .mtz fil | | [more .mtz from DIFFLAUE] | | | | DIFFLMRG .mtz file | | (shelx file) | | | .mtz file | Reflection data | eg h k l F sig(F) Reflection data for difference Fouriers: h k l F1 F2 phase PREDICTION OF THE LAUE PATTERNS The positions of the spots in a Laue pattern may be predicted given the cell dimensions, the crystal setting, the wavelength range lambda-min to lambda-max and the minimum observable plane spacing, dmin. In a prediction, it is assumed that lambda-min, lambda-max and dmin are sharp limits whereas in reality usually 'soft' limits. The programs LAUEGEN and NEWLAUE may be used for making such predictions. LAUEGEN is used on an X-windows display and allows for colour coded plots and also black and white plots where sliders can be used to examine the effects on the predicted pattern of changing the soft limits. NEWLAUE is an older program and uses a terminal which supports T4010 graphics. DETERMINATION OF THE CRYSTAL ORIENTATION When processing a set of Laue data it is necessary to determine the crystal orientation. An auto-indexing method (from M. Elder) enables this to be done when the unit cell and crystal to detector distance are known. The procedure involves matching the observed angles between the reciprocal lattice vectors for a selection of nodal reflections with a pre-calculated table of angles of potential nodal relections. The orientation is defined in terms of missetting angles from a standard crystal setting. The method is available in the programs LAUEGEN and the older NEWLAUE program. PARAMETER REFINEMENT Before accurate intensity measurement can be attempted, it is essential to get a very close match between the predicted and observed reflection positions. This may be done by either the program LAUEGEN (for an X-windows display) or the older program GENLAUE (for a T4010 compatible terminal). These programs allow for pattern prediction image display (threshold plot when usung GENLAUE) and parameter refinement. The parameters refined by the program are the position of the diffraction pattern centre, the crystal to detector distance, the missetting angles and, optionally, the cell parameters. LAUEGEN also allows for the determination and refinement of distortion parameters.The refinement seeks to minimise the difference between the observed and calculated positions. The best spot positions to use are the spatially isolated nodal spots. INTENSITY MEASUREMENT The problems of obtaining background corrected intensity measurements from Laue data are very similar to those experienced when using other detector geometries. For proteins, however, there may be additional problems as the spots, especially along lunes of data, may be very closely spaced. An LDM based integration program INTLDM is now available and an equivalent integration option is now also available in the LAUEGEN program. All the integration programs will handle radially streaked (elliptical) spots and can deconvolute intensities from spatially overlapped spots. INTLAUE was written by T.J. Greenhough and A.K. Shrive and was based originally on the oscillation film processing program. The program can allow for the refinement of parameters, such as twist, tilt and bulge (or twist, tilt, roff, toff for the MAR image plate), based on the the positions of well separated nodal reflections if these have not already been determined in LAUEGEN. It has options are available for both box integration and for profile fitting. The program has procedures for the deconvolution of spatially overlapping spots and can allow for varying spot length as a function of angle around the pattern centre. SCALING WITHIN (FILM) PACKS Inter-plate scaling factors of the form I1 = I2*exp(a*lambda**3) have been assumed. The scaling may be done as part of the overall scaling procedure using the program LSCALE or using the stand alone program AFSCALE . For data recorded on film, three wavelength ranges are normally needed(lambda <= 0.49, 0.49 <= lambda <= 0.92, lambda > 0.92) because of the discontinuities in the scaling curve at the Silver and Bromine k edges, 0.49 and 0.92 A. The coefficient 'a' is evaluated for each range and refined using an iterative process. WAVELENGTH NORMALISATION The program LSCALE or the older program LAUENORM is used to derive a wavelength normalisation curve based on symmetry equivalent reflections measured at different wavelengths. The scaling may be divided into wavelength ranges to allow for allow for absorption discontinuities in the scaling function. An alternative method, which may be appropriate in some cases, uses the program LSCALE or the older program LAUESCALE and involves scaling against a reference (e.g. monochromatic) set of data. ABSORPTION CORRECTION The program LSCALE can carry out a position and wavelength dependent absorption correction making use of symmetry equivalent reflections. A position only absorption correction is also available in the older LAUESCALE program. UNSCRAMBLING HARMONICS A complication of the Laue method is the presence of harmonics which are superimposed to form composite reflections on the image (multiples). The intensity of such a reflection is the the sum of the intensities of the constituent reflections. The integrated intensities of these multiples are evaluated as for the other reflections. The deconvolution may be done by the program LSCALE (or LAUENORM . These use the multiple spots and symmetry equivalent reflections recorded at different wavelengths to deconvolute the constituent reflection intensities. An alternative method depends on the fact that different wavelengths are absorbed differently by the plates (films) and by any metal foils placed between them. The program UNSCRAM may be used to carry out this unscrambling process. Such data, if present, will also be used in the deconvolution process in LSCALE. A method for deconvoluting Laue harmonics data using direct methods in real space is also available (DECONV) . PROGRAMS FOR PROCESSING DIFFERENCE DATA The programs DIFFLAUE or LAUEDIFF are used to prepare difference data scaled on an individual reflection basis to a reference set of data. The coefficients are calculated as: (F(Laue_time2) - F(Laue_time1)) Fdiff = ------------------------------- x F(Reference_time1) F(Laue_time1) The reference e.g. native set of data could be a set of monochromatic data or a set of Laue data processed as described above and using the LSCALE or LAUENORM wavelength normalisation procedure. Phases in the reference data set may be passed on to the output set of data containing the difference coefficients. The preferred method of the two available is to use DIFFLAUE in which individual pairs of plates, one from each pack, are scaled together and where the data from each of the pairs of plates are merged as the final processing stage. In the alternative method, using LAUEDIFF (not shown on the flowchart), the plate scaling within a pack is done using AFSCALE. LAUEDIFF then scales together the two packs and calculates the difference data as indicated above. It is important to remember that the crystal orientation must be very close for the two exposures so that the wavelength associated with each reflection is essentially the same (say within 0.1 Angstrom) for both measurements as the wavelength normalisation is effectively done on a reflection by reflection basis. If several packs of data are processed using DIFFLAUE, the resultant data sets may be merged using the program DIFFLMRG . REFERENCES 1) Campbell, J.W., Clifton, I.J., Elder, M., Machin, P.A., Zurek, S., Helliwell, J.R., Habash, J., Hajdu, J. and Harding, M.M. (1987) in Springer series in Biophysics, Vol 2., Biophysics and Synchrotron Radiation (ed Bianconi, A. and Congiu Castellano, A.),(Springer Verlag, Berlin, Heidelberg, New York, London, Tokyo) pp. 52-60 2) Campbell J.W., (1994a) "XDL_VIEW, an X-windows based Toolkit for Crystallographic and Other Applications" (1995), J. Appl. Cryst., 28236-242 3) Campbell J.W., (1994b) "LAUEGEN, an X-windows based Program for the Processing of Laue X-ray Diffraction Data" (1995) J. Appl. Cryst., 28228-236 4) Campbell J.W., Clifton I.J., Harding M.M. and Hao Q. (1994) "A Laue Data Module (LDM) for use in the processing of Laue X-ray Diffraction Data" (1995) J. Appl. Cryst., 28635-640 5) Greenhough T.J & Shrive A.K., (1994); J. Appl. Cryst., 27111-121 6) Helliwell, J.R., Habash, J., Cruikshank, D.W.J., Harding, M.M., Greenhough, T.J., Campbell, J.W., Clifton, I.J., Elder, M., Machin, P.A., Papiz, M.Z. and Zurek, S. (1989) J. Appl. Cryst., 22483-497. 7) Arndt, U.W. & Wonacott, A.J., (19;77). "The Rotation Method in Crystallography" Amsterdam: Netherlands. 8) Wonacott, A.J. (1980) Notes on a suite of programs for processing oscillation camera film data. 9) Campbell, J.W., Hao, Q., Harding, M.M., Nguti N.D. and Wilkinson, C. "LAUEGEN version 6.0 and INTLDM" J. Appl. Cryst. (1998) 31, 496-502 10) Arzt, S, Campbell, J.W., Harding, M.M., Hao, Q. and Helliwell J.R., "LSCALE - The New Normalisation, Scaling and Absorption Correction Program in the Daresbury Laue Software Suite" J.Appl. Cryst. (1999) in press Figure 1 Flowchart of Main Processing Sequence