Data Processing     HKL2000      Mosflm
Data collection and            processing•   mount crystal•   evaluate quality    •   diffraction quality, resolution, spl...
Image from R-axis IV            detector•   detector is square,    so the corners are    higher resolution;    the main li...
Bad images• Always check crystals at 0° and 90° to check  for splitting, large diffuse reflections, doublets               ...
• Zoom in enough,  intensity value of  each pixel is  displayed
Peak searchPeak SearchAsk theprogram tosearch theimage forreflections.Accept theFound reflectionsThis writes “peaks.file”
Space group predictionThe program               Volume of the primitive cell   559745.determines the best       Lattice   ...
Show predicted spots• Are there spots  under the  predictions?• Are there spots  which are not  predicted?• What are the r...
use zoomwindow and  ‘removepred’ key to    togglepredictions
Reflection profiling• spots are assumed to be Gaussian in shape• if center is ‘maxed out’, then the curve is  flattened on to...
autoindex unit cell   157.23    80.46    104.06     90.00   121.75   90.00                        crystal rotx, roty, rotz...
Mosaicity Mosaicity is a measure of how wide a spot is in phi (rotation angle).          Mos=1.0!Note that here the image ...
Change mosaicity between refinement “go’s” and refine again.              Check predictions for reflection coverage       ...
Averaged spot profile in sector   2, 3 (x,y) # of spots     123                                   Weighted position of the...
HKL’s assigned• The process of integration causes a series of “.x” files to be  written which contains the indexed reflect...
Check 90° awayDon’t forget to goback and checkanother image atleast 45-90! awayfrom the first image.Again check imagefor s...
Scaling•   a program that merges the .x files•   scale images together to account for absportion effects    and crystal dec...
Redundancy and       completenessMost reflections in this data set are collected at least 7times. The higher the redundancy...
I/σI and completenessWe limit the resolution based on two factors:1. when the % reflections is above 50% for I/σI=2in the e...
I/σI and completenessWe limit the resolution based on two factors:2. Rmerge goes above ∼50%in the example above, Rmerge ne...
Data processing Lab Lecture
Data processing Lab Lecture
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Data processing Lab Lecture

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  • Data processing Lab Lecture

    1. 1. Data Processing HKL2000 Mosflm
    2. 2. Data collection and processing• mount crystal• evaluate quality • diffraction quality, resolution, splitting, cryo-protection• Auto-index • space group, cell dimensions, strategy for collection, mosaicity• integrate a series of images• scale images together • completeness, Rmerge, systematic absences, I/σI
    3. 3. Image from R-axis IV detector• detector is square, so the corners are higher resolution; the main limit of resolution is the radius along the vertical and horizontal axes• backstop appears as a white shadow
    4. 4. Bad images• Always check crystals at 0° and 90° to check for splitting, large diffuse reflections, doublets 90° - the splitting is obvious 0° - image doesn’t diffract very in the forms of wide, diffuse high, but spots are well formed spots
    5. 5. • Zoom in enough, intensity value of each pixel is displayed
    6. 6. Peak searchPeak SearchAsk theprogram tosearch theimage forreflections.Accept theFound reflectionsThis writes “peaks.file”
    7. 7. Space group predictionThe program Volume of the primitive cell 559745.determines the best Lattice Metric tensor distortion index Best cell (symmetrized) Best cell (without symmetry restrains)lattice fit to each primitive cubic 17.70% 88.21 80.46 100.09 66.31 66.75 63.03 89.59 89.59 89.59 90.00 90.00 90.00point group. It prints I centred cubic 11.58% 100.09 88.41 131.41 95.23 104.52 91.61 106.64 106.64 106.64 90.00 90.00 90.00the closest real fit to F centred cubic 7.18% 135.40 131.67 131.41 75.42 82.77 82.91 132.83 132.83 132.83 90.00 90.00 90.00the space group primitive rhombohedral 2.91% 100.09 100.10 104.06 51.28 51.15 47.40restrictions and then 101.42 101.42 101.42 49.94 49.94 49.94 84.33 84.33 265.57 90.00 90.00 120.00(in a second line)what primitive hexagonal 12.90% 80.46 88.21 100.10 88.27 66.30 116.97 84.33 84.33 100.10 90.00 90.00 120.00the space groups primitive tetragonal 16.77% 88.21 80.46 100.09 66.31 66.75 63.03 84.33 84.33 100.09 90.00 90.00 90.00restrictions would I centred tetragonal 7.05% 80.46 104.06 135.40 80.93 90.10 89.88 92.26 92.26 135.40 90.00 90.00 90.00actually demand. The primitive orthorhombic 16.58% 80.46 88.21 100.09 66.75 113.69 116.97tensor index gives a 80.46 88.21 100.09 90.00 90.00 90.00 C centred orthorhombic 12.45% 80.46 157.23 100.10 76.14 66.30 89.84correlation of the fit. I centred orthorhombic 80.46 157.23 100.10 90.00 90.00 90.00 3.76% 80.46 104.06 135.40 80.93 90.10 89.88 80.46 104.06 135.40 90.00 90.00 90.00 F centred orthorhombic 6.36% 80.46 157.23 183.31 74.91 90.00 89.84 80.46 157.23 183.31 90.00 90.00 90.00 primitive monoclinic 12.46% 80.46 100.09 88.21 113.25 116.97 66.31 80.46 100.09 88.21 90.00 116.97 90.00 C centred monoclinic 0.07% 157.23 80.46 104.06 89.88 121.75 90.16 157.23 80.46 104.06 90.00 121.75 90.00 primitive triclinic 0.00% 80.46 88.21 100.09 66.75 66.31 63.03 autoindex unit cell 80.46 88.21 100.09 66.75 66.31 63.03 crystal rotx, roty, rotz -71.263 116.160 -73.145 Autoindex Xbeam, Ybeam 118.97 119.89
    8. 8. Show predicted spots• Are there spots under the predictions?• Are there spots which are not predicted?• What are the red spots?
    9. 9. use zoomwindow and ‘removepred’ key to togglepredictions
    10. 10. Reflection profiling• spots are assumed to be Gaussian in shape• if center is ‘maxed out’, then the curve is flattened on top, and the intensity cannot be determined accurately
    11. 11. autoindex unit cell 157.23 80.46 104.06 90.00 121.75 90.00 crystal rotx, roty, rotz 169.439 -13.341 -22.651 Autoindex Xbeam, Ybeam 118.97 119.89 position 245 chi**2 x 1.41 y 2.42 pred. decrease: 0.000 * 245 = 0.0 partiality 245 chi**2 0.24 pred. decrease: 0.000 * 245 = 0.0 position 852 chi**2 x 2.36 y 1.59 pred. decrease: 0.001 * 852 = 0.5 partiality 2788 chi**2 0.90 pred. decrease: 0.000 * 2788 = 0.1 CrysZ (beam) -22.673 shift 0.012 error 0.019 CrysY (vertical) -13.291 shift -0.054 error 0.013 CrysX (spindle) 169.490 shift 0.046 error 0.011 Cell, a 157.86 b 80.43 c 104.41 alpha 90.00 beta 122.00 gamma 90.00 shifts 0.63 -0.03 0.35 0.25 errors 0.12 0.03 0.04 0.04Watch chi**2 values CassY (vertical) -0.079 shift 0.001 error 0.043decrease or level out CassX (spindle) X beam 0.008 shift 119.001 shift -0.002 error 0.027 error 0.039 0.011 Y beam 119.930 shift 0.039 error 0.009 Radial offset 0.012 shift 0.000 error 0.021 Angular offset -0.061 shift -0.005 error 0.022 Crossfire y -0.001 shift -0.023 error 0.012 Crossfire x 0.022 shift 0.022 error 0.014 Crossfire xy 0.020 shift -0.001 error 0.018 Watch value on end of first line. This is the value of the predicted difference if you were to refine again. At ‘zero’ additional refinement is not as useful.
    12. 12. Mosaicity Mosaicity is a measure of how wide a spot is in phi (rotation angle). Mos=1.0!Note that here the image isfrom a 0.5 ! rotation. Thisis less than the mosaicity.Only partial reflections arerecorded.
    13. 13. Change mosaicity between refinement “go’s” and refine again. Check predictions for reflection coverage Playing with Mosaicity The Mosaicity directly effects the rotation angle during data collection. We want to avoidoverlaps (red). If the mosaicity is less than the rotation angle then full reflections are recorded, if the rotation angle is less than the mosaicity then all reflections are only partially recorded. Mos=2.0! Mos=0.5!
    14. 14. Averaged spot profile in sector 2, 3 (x,y) # of spots 123 Weighted position of the spots 121.111, 173.574 (x,y) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . . . . . . . . . . . . . . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . . . . . . . . . . . . . . . . . . . . . . .As the image is processed, spot 0 0 0 0 0 0 0 0 0 0 . . . . . . . . . . 0 0 0 0 0 0 0 0 . . . . . . . . 0 . 0 . 0 . 0 . 0 .profiles are printed. These are 0 0 0 0 0 0 0 0 0 0 . . . . . . . . . . 0 0 0 0 0 0 0 0 . . . . . . . . 0 . 0 . 0 . 0 . 0 .two dimensions profiles of the 0 0 0 0 0 0 0 0 0 0 . . . . . . . . . . 0 0 0 0 0 0 0 0 . . . . . . . . 0 . 0 . 0 . 0 . 0 .three dimensional reflections. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . . . . . . . . . . . . . . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 . . . . . . . . . . - - - . . . . . . . . . . 0 0 0 0 0 0 0 1 1 1 1 2 2 2 1 1 1 0 0 0 0 0 0Background should be 0, the . . . . . . . . - + 0 0 0 0 0 0 1 1 2 2 + + + + - . . . 3 5 6 6 4 2 1 1 . 0 . 0 . 0 . 0 . 0spots are normalized in these . . . . . . . - + + 0 0 0 0 0 1 1 2 3 6 + + + + + - . . 13 20 27 22 10 3 1 1 . 0 . 0 . 0 . 0 . 0plots and center should be . . . . . . . + + + 0 0 0 0 0 1 2 3 7 17 + + + + + + . . 36 58 62 47 19 5 2 1 . 0 . 0 . 0 . 0 . 0 . . . . . . - + + + + + + + + + - . . . . . .<100. 0 0 0 0 1 1 2 5 13 29 . . . . . . - + + + 52 68 64 43 18 5 2 1 + + + + + + - . 0 . 0 . 0 . 0 . 0 . 0 0 0 0 1 1 3 6 14 26 37 39 32 20 9 3 1 1 0 0 0 0 0 . . . . . . - + + + + + + + + + - . . . . . .+/- indicates type of pixel 0 0 0 0 0 1 2 6 10 14 . . . . . . . + + + 16 15 11 7 4 2 1 0 + + + + + + . . 0 . 0 . 0 . 0 . 0 . background “.” 0 0 0 0 0 1 2 3 5 6 . . . . . . . - + + 6 5 4 3 2 1 1 0 + + + + + - . . 0 . 0 . 0 . 0 . 0 . 0 0 0 0 0 1 1 1 2 2 2 2 1 1 1 0 0 0 0 0 0 0 0 overlap “-” or . . . . . . . . - + + + + + - . . . . . . . . 0 0 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 actual spot “+” . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 - - - . . . . . 0 0 0 0 0 0 0 0 . 0 . 0 . 0 . 0 . 0Determined by size of . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 . . . . . . . . 0 0 0 0 0 0 0 0 . 0 . 0 . 0 . 0 . 0integration boxes listed in . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 . . . . . . . . 0 0 0 0 0 0 0 0 . 0 . 0 . 0 . 0 . 0 . . . . . . . . . . . . . . . . . . . . . . .auto.dat. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 . . . . . . . . . . . . . . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 . . . . . . . . . . . . . . . . . . . . . . .
    15. 15. HKL’s assigned• The process of integration causes a series of “.x” files to be written which contains the indexed reflections. HEADER 1 met8p -0.00465175 0.00519329 -0.01006241 0.676244 -0.138237 -0.723592 0.00294147 0.01116484 0.00423633 -0.622640 0.417704 -0.661697 0.00505222 -0.00171869 -0.00288537 0.393718 0.898005 0.196398 0.00000 0.50000 1866.66663 0.84450 -22.6727 -13.2914 169.4859 1.2000-1-1-1-1-1-1 -25 21 -23 1 -450.2 -558.9 0.47-541.6 0.921 9.4 908.1 1.196 1950.8 0.156 0.1 -26 18 -24 1 646.4 700.2 0.58 753.5 0.923 12.9 838.2 1.190 3223.0 0.278 0.1 -25 19 -24 1 -1066.9 -1009.3 0.48 748.2 0.922 14.0 862.2 1.188 3192.6 0.605 0.3 -24 20 -24 1 958.1 1300.8 0.42 761.5 0.922 15.0 886.1 1.187 3059.5 0.186 0.4 -28 14 -25 1 335.9 684.0 0.48 738.6 0.923 15.1 744.4 1.186 3439.4 0.188 0.1 -27 15 -25 1 981.8 423.1 0.37 746.0 0.923 16.3 768.6 1.185 3437.6 0.573 0.2 -30 10 -26 1 3987.9 5029.6 0.54-678.7 0.921 17.1 650.4 1.184 2562.2 0.461 0.2 -26 16 -25 1 251.6 -181.3 0.51 739.9 0.923 17.4 792.7 1.183 3693.9 0.466 0.3 -29 11 -26 1 1160.3 1373.3 0.47 779.6 0.922 18.3 674.9 1.182 3512.9 0.601 0.3 -25 17 -25 1 -1021.2 -951.7 0.48 740.6 0.924 18.4 816.8 1.181 3512.8 0.045 0.4 -28 12 -26 1 1858.6 2942.4 0.54 745.8 0.923 19.6 699.3 1.180 3865.9 0.382 0.3 -27 13 -26 1 -360.6 -1069.0 0.50 735.9 0.923 20.6 723.5 1.178 3929.4 0.049 0.4 -23 23 -22 1 -515.1 -319.2 0.41 725.6 0.922 27.2 963.4 1.168 3136.4 0.467 0.2 -22 24 -22 1 737.4 83.7 0.44-754.9 0.921 28.3 987.1 1.167 2859.0 0.414 0.3 -24 20 -23 1 678.0 880.1 0.42 707.3 0.925 30.8 893.5 1.162 4553.3 0.448 0.2 -23 21 -23 1 890.2 794.5 0.39 704.6 0.924 31.9 917.3 1.160 4548.9 0.496 0.3 h, k, l, xpos, ypos, fract., background, overlap, and spot intensity.
    16. 16. Check 90° awayDon’t forget to goback and checkanother image atleast 45-90! awayfrom the first image.Again check imagefor splitting. Spotprofile etc.
    17. 17. Scaling• a program that merges the .x files• scale images together to account for absportion effects and crystal decay• compare reflections measured more than once and determine mean and standard deviation• calculate completeness, Rmerge, I/σI, and error probabilities• reject outliers• determine final space group by viewing systematic absences• output a merged file of reflections for use in structure determination and refinement
    18. 18. Redundancy and completenessMost reflections in this data set are collected at least 7times. The higher the redundancy the more accuratethe data. Redundancy is a function of the space groupand the number of degrees of data collected
    19. 19. I/σI and completenessWe limit the resolution based on two factors:1. when the % reflections is above 50% for I/σI=2in the example above, it’s only 28.4% at I/σI=2 in the highest resolutionshell, so data should have been collected to a higher resolution
    20. 20. I/σI and completenessWe limit the resolution based on two factors:2. Rmerge goes above ∼50%in the example above, Rmerge never goes above 50% in the highestresolution shell, so again data should have been collected to a higherresolutionFinal statistics are usually reported for the overall value and the valuefor the highest resolution shell
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