The document discusses the process of data collection and processing for X-ray crystallography. This includes mounting the crystal, evaluating diffraction quality, auto-indexing to determine unit cell dimensions and space group, integrating images, scaling images together, and checking for issues like splitting or diffuse reflections in the images. It also discusses optimizing parameters like mosaicity during refinement to ensure full rather than partial reflections are recorded. Spot profiles are averaged and printed to analyze reflection shapes.

1. Data Processing
HKL2000
Mosflm

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. 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. 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. • Zoom in enough,
intensity value of
each pixel is
displayed

6. Peak search
Peak Search
Ask the
program to
search the
image for
reflections.
Accept the
Found reflections
This writes “peaks.file”

7. Space group prediction
The 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 90.00
point 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 90.00
the 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 90.00
the space group primitive rhombohedral 2.91% 100.09 100.10 104.06 51.28 51.15 47.40
restrictions 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 90.00 120.00
the 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 90.00
restrictions 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 90.00
actually demand. The primitive orthorhombic 16.58% 80.46 88.21 100.09 66.75 113.69 116.97
tensor 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 89.84
correlation 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. Show predicted spots
• Are there spots
under the
predictions?
• Are there spots
which are not
predicted?
• What are the red
spots?

9. use zoom
window and
‘remove
pred’ key to
toggle
predictions

12. 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

13. 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 0.04
Watch chi**2 values CassY (vertical) -0.079 shift 0.001 error 0.043
decrease 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.

14. Mosaicity
Mosaicity is a
measure of how
wide a spot is
in phi (rotation
angle).
Mos=1.0!
Note that here the image is
from a 0.5 ! rotation. This
is less than the mosaicity.
Only partial reflections are
recorded.

15. 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 avoid
overlaps (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!

16. 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 0
Background 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
.
0
spots 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
.
0
plots 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
.
0
Determined 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
.
0
integration 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
. . . . . . . . . . . . . . . . . . . . . . .

17. 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.

18. Check 90° away
Don’t forget to go
back and check
another image at
least 45-90! away
from the first image.
Again check image
for splitting. Spot
profile etc.

19. 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

20. Redundancy and
completeness
Most reflections in this data set are collected at least 7
times. The higher the redundancy the more accurate
the data. Redundancy is a function of the space group
and the number of degrees of data collected

21. I/σI and completeness
We limit the resolution based on two factors:
1. when the % reflections is above 50% for I/σI=2
in the example above, it’s only 28.4% at I/σI=2 in the highest resolution
shell, so data should have been collected to a higher resolution

22. I/σI and completeness
We limit the resolution based on two factors:
2. Rmerge goes above ∼50%
in the example above, Rmerge never goes above 50% in the highest
resolution shell, so again data should have been collected to a higher
resolution
Final statistics are usually reported for the overall value and the value
for the highest resolution shell