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The first step in data
collection is always to mount a crystal and take a diffraction pattern.
Often a still exposure is taken first, followed by an oscillation if the
still looks promising. What can we conclude from the image shown in
Figure 1? How about Figure 2? (Click on the figure for an enlarged
view.)
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| Figure 1 |
Figure 2 |
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From the
image alone several things can be checked: |
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Singleness of crystal
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anything other than a single
pattern of well-defined lunes probably indicates a split, multiple, or
twinned crystal. Translating the crystal along the spindle may allow
finding a region which is single. |
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Mosaicity |
more spots than expected for
the oscillation range are produced if the mosaic spread of the crystal
is high. From the image itself one can get some feel for mosaicity, but
this should be checked after indexing it (see below). |
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Overloads |
saturated pixels are
distinguished from others on the displays available at the stations:
they appear in color on the Fuji scanner Image_Analyze display and in
yellow on the CCD display. There should be no more than a few percent
overloaded reflections in the resolution range of interest. To collect a
wide range of intensities, it may be necessary to take multiple passes
through the total oscillation range, with different exposure times. |
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Shadowing |
it is possible for equipment
such as a cooling nozzle to block part of the detector surface. This is
usually obvious, but not always. In the case of a short exposure with
relatively few spots (from a small molecule crystal, for example), one
may need to look closely to detect the region where data are missing.
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Spot separation |
successful integration of
reflections requires enough separation between them. Each panel of
Figure 3 shows a small region of a diffraction pattern, containing a row
of spots. A plot of pixel values along the horizontal dashed line is
superimposed on each display. The required distance between spots
depends on the spot size, but is typically about 10 pixels, as in the
example of Figure 3a. The 6-pixel separation in Figure 3b. will clearly
cause difficulty in integration and should be avoided if possible,
either by moving the detector back or by narrowing the oscillation
range. |
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Signal-to-noise |
adequate peak to background
ratio is needed for good data. Scaling by the image display program may
make an image look fine when in fact it is not. A check of the
background values may reveal the problem; backgrounds over about 1000
for image plates or 5000 for CCD's are suspicious. A program is being
developed to give plots of background and signal-to-noise as a function
of resolution, to aid in this aspect of image evaluation. |
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| Figure 3a |
Figure 3b |
For the two images shown,
both crystals appear to be single. Although not shown in these figures,
neither had excessive overloads in the resolution range of interest. A
shadowed region is visible in Figure 2, but only a small fraction of the
data will be obscured by it. The spot separation is close but adequate
in Figure 1. Figure 4, however, reveals a problem. Here is plotted the
background (and a few peaks) along a radius for the Figure 1 (lower
trace) and Figure 2 (upper trace) images. The background in Figure 2 is
clearly excessive, and will result in poor signal-to-noise for the data
from this image. This high background is probably due to scattering from
frozen solvent, either in or surrounding the crystal. It would be
advisable to look for a better crystal, or to try mounting in a smaller
loop if external solvent is the problem.
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| Figure 4. Plot along a
radial line for Figure 1 (lower) and Figure 2 (upper) images. |
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Once a visually
satisfactory image has been obtained, the crystal should be rotated,
usually by 90 degrees, and another exposure taken, to check for
anisotropic mosaicity, splitting that was not apparent on the first
image, and any crystal centering problem. The latter is probable when
diffraction is very weak or absent at the second spindle position but is
fine on a repeat of the first exposure.
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If the second
image is good, it is time to index an image. This is easily done using,
for example, HKL-2000 or Denzo (part of the HKL program package, by Z.
Otwinowski and W. Minor); the only parameters needed are the direct beam
position and the crystal-to-detector distance. A successful indexing
produces the result shown in Figure 5a. The predicted reflections, shown
as green, yellow, and red circles, fall on or almost on the actual
spots, and very few spots have no corresponding predictions. The
predictions in Figure 5b were produced when an incorrect
crystal-to-detector distance was supplied. This is the most common cause
of a bad indexing. It is largely due to the difficulty of reading an
accurate distance on the MacCHESS oscillation cameras; a new camera
design will remedy this problem. A distance error makes all the
calculated cell dimensions too high or too low; if the correct values
are known it is easy to adjust the distance until the calculated values
are reasonable. If the distance and direct beam position are correct,
and the image has at least a few dozen good spots, the indexing should
succeed. If not, the crystal may be twinned, so that the spots are not
from a single lattice.
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Figures 5a
(left) and 5b (right), showing portions of a diffraction image (in
shades of gray) with predicted reflection positions superimposed
(colored circles: green for fully recorded reflections, yellow for
partials, red for "problem" reflections). Display from the HKL package.
Click on the figure for an enlarged view.
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| Figure 5a |
Figure 5b |
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Once a predicted diffraction pattern has
been generated, crystal mosaicity may be evaluated. If the predicted
lunes are too narrow, the mosaicity is higher than the value supplied to
Denzo (assuming the correct oscillation range). The mosaicity may
readily be varied until the prediction matches the real pattern. If
spots appear inbetween the predicted lunes and no value for mosaicity
will account for them, the crystal is probably twinned, or has a
satellite. There is no point in wasting time on such a crystal unless
the extra spots are very few or all the crystals of the material are
equally bad.
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The appropriate
oscillation range may be determined by making predictions for various
ranges and checking for overlapping reflections. Using a mosaicity a
little on the high side for safety, a range that is as wide as possible
without generating more than a few overlaps may be selected. In some
cases a narrower range than this may be desired, for the reason of
reducing background. A few more test exposures may be needed to settle
the question. If the unit cell dimensions are not all quite similar,
predictions should be made for several spindle settings, as different
oscillation ranges may be appropriate at different crystal orientations.
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The ideal data set is 100%
complete, with most reflections measured several times. Naturally, this
is not always possible. From an indexed image, however, it may be
determined how much of the unique data can be collected on the crystal,
and what range of spindle angles must be covered to get this fraction. A
program now being developed at MacCHESS, "m.simulate", will aid in this
determination.
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The fraction of reciprocal
space that must be covered to collect all the unique data to a given
resolution depends on the crystal symmetry and on whether anomalous data
are required. It is common to say that because a crystal is monoclinic,
one must collect 180 degrees of data, or because it is tetragonal one
only needs 45 degrees. In fact, the rotation range needed to collect the
unique data depends on the orientation of the rotation axis relative to
the unit cell axes, i.e. on the orientation of the crystal on the
camera. In the real world, an additional factor is introduced by the
limited area of the detector. For the CCD detectors in particular,
recording resolved spots to high resolution may require offsetting the
detector perpendicular to the x-ray beam. This results in some
combination of a loss of redundancy and a loss of unique data for a
given rotation range. The table below illustrates the effects of varying
crystal orientation and detector position. This is a case where some
data are off the edge of the detector if it is not offset, so that with
the CCD centered even a 180 degree rotation of the crystal only gives
about 90% of the unique data at best. If the crystal is aligned with c*
along the spindle, only 90 degrees of rotation are needed to give the
maximum completeness, but this maximum is only 76%. If the detector is
offset, a complete data set may be obtained, but it requires taking a
full 360 degrees of data if anomalous data are needed or if the crystal
orientation is not optimum.
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In this table, the three
crystal orientations shown are: 1) c* along the spindle axis, x-ray beam
along b* at spindle angle 0 (Denzo crystal rotation angles rotx = roty =
rotz = 0); 2) b* along the spindle axis, x-ray beam along c* at spindle
angle 0 (Denzo crystal rotation angles rotx = rotz = 0, roty = 90); 3) a
general orientation, Denzo crystal rotation angles rotx = 10, roty = 30,
rotz = 20. "% unique" gives the percentage of the unique reflections,
ignoring anomalous dispersion, that could be recorded from the crystal
by rotating it over the given range of spindle angles. "% anom" gives
the percentage of anomalous pairs (Bijvoet mates) that would be recorded
during the same rotation. "Redundancy" gives the average number of
symmetry-related observations of each unique reflection that would be
recorded, assuming that anomalous data are not needed. The redundancy of
anomalous measurements (not shown) would be lower. These percentages
take no account of losses due to overloaded or overlapping reflections.
Data for table from m.simulate program.
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Orienting a crystal with a
symmetry axis along the x-ray beam can serve to minimize the rotation
range required to collect a nearly complete data set. Alternatively,
measurement of anomalous data may be facilitated by orienting the
crystal to put Bijvoet pairs on each image. In the case of a unit cell
with one long axis, placing that axis along the spindle allows wider
oscillations to be taken than otherwise. The advantages of using an
oriented crystal must be considered in light of the difficulty in
scaling frames from a rotation series on such a crystal together,
particularly in the lower symmetry classes. A data set from a second,
differently oriented, crystal will probably resolve this problem. An
additional consideration is that, for some symmetries, data collected by
rotation about a symmetry axis will be incomplete no matter how many
degrees of rotation are taken, due to the "missing cone" problem.
Limitations in detector area may also become more important for oriented
crystals. Figure 6, drawn by the program Geomview (from The Geometry
Center at the University of Minnesota), shows the fraction of unique
reflections collected in a 360 degree rotation of a small molecule
crystal. The figure represents a portion of reciprocal space: the blue
surface encloses the total unique volume (to the limiting resolution of
the crystal) for this orthorhombic cell; the magenta surface encloses
the points corresponding to the unique reflections which were actually
measured. Along the left-hand edge, the magenta surface is just inside
the blue, showing complete coverage, but at the lower right a
substantial number of the unique reflections were not collected. The
crystal was oriented with b* near, but not on, the spindle axis; the CCD
detector was offset, in order to get the desired resolution. The missing
regions are due to a combination of limited detector size, "missing
cone" effect, and a cooling nozzle shadow that was not obvious during
data collection (due to the small number of spots per image). Although
this image was generated using the reflections actually collected, the
missing regions due to crystal orientation and detector geometry could
have been predicted ahead of time using m.simulate, and the desirability
of taking more data on a second, differently oriented, crystal would
have been clear. In future, users will be able to check the potential
completeness of their data before taking it. An additional capability
planned for m.simulate is that of reading in an earlier data set and
telling whether the current crystal will fill in gaps or merely
replicate earlier data.
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Figure 6
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At CHESS, considerations of
desirable crystal orientations are currently moot, as reorienting of
crystals is limited to what can be done on the goniometer arcs. This may
change in the future, however, and it is sometimes possible to influence
a crystal's orientation during the mounting process.
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To optimize the anomalous
signal from a crystal not oriented with a mirror plane perpendicular to
the spindle, it may be desirable to use the "inverse beam" approach:
after a few degrees of data have been taken the crystal is rotated 180
degrees and the same amount of data collected. The second set of images
will contain the anomalous mates of reflections on the first set. Note
that this will only be true for all reflections if the detector is
centered.
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Enough
information is now available to determine the experimental parameters
for data collection.
These are: |
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Exposure time
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set to give few overloads in
the resolution range of interest and a reasonably low background.
Multiple passes with different exposure times may be necessary to get a
wide resolution range. The minimum exposure time per degree is set by
the maximum speed of the spindle motor. For strongly diffracting
crystals, it may be necessary to attenuate the x-ray beam to avoid
overloading. |
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Oscillation range
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set to minimize number of
exposures, while allowing few overlapping reflections and keeping
background low. May vary with spindle setting. |
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Detector distance and offset
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set to avoid having spots
too close, while collecting data as close to the limiting resolution of
the crystal as possible. |
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Limits of total oscillation
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set by range needed to get
the most complete data set possible for crystal's orientation. More than
the minimum range may be taken if high redundancy is wanted. |
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Crystal orientation
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set, if desired and
possible, to minimize number of exposures or maximize quality of
anomalous data. Except for rotation about the spindle, can only be
controlled (at CHESS, now) to a limited degree, and would usually not be
changed. |
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Short-lived crystals |
The foregoing describes an
approach of careful checking before starting data collection. This is
appropriate when the crystal on the camera is frozen (as is now standard
at CHESS) and will not be harmed by waiting for the 10-15 minutes it
takes for full evaluation and planning. When the crystal is not frozen,
or is subject to rapid decay even when frozen, it is better to just make
a quick examination of an image for crystal splitting, spot overlaps,
etc., and proceed directly to data collection. Then, while the next
crystal is being mounted, the images just taken can be examined for
mosaicity, etc. and parameters adjusted for other crystals in the batch.
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Multiwavelength data
collection |
The efficient collection of
data at multiple wavelengths, for MAD phasing, involves the same
considerations as the monochromatic case. Aside from changing
wavelengths between exposures and periodically taking energy scans, the
data collection process itself is the same, and the same criteria are
used to select good crystals, set the oscillation range, and so forth.
Because of the importance of Bijvoet pairs in MAD phasing, it is
necessary to take particular care with crystal orientation and detector
offset, and data may be collected using the "inverse beam" approach. See
information on
"Planning MAD data collection" for more
detail. In addition, the extra time needed for collecting each rotation
range three or four times makes it especially important to optimize all
experimental parameters, if the data set is to be completed in the time
available.
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Unusual data collection
modes |
For very weakly diffracting
crystals, long exposures, on the order of an hour, may be required. If
normal oscillation exposures are taken and the beam dumps halfway
through one of them, the image will probably be too weak to be useful. A
"long-exposure" mode is available, in which only one pass is taken
through the oscillation range, with the spindle rotating in a series of
small steps rather than continuously. In order to compensate for
variation in beam intensity with time, exposure at each position is for
a fixed number of counts, not a fixed time. With this mode, if an
exposure is terminated prematurely one has a narrower oscillation range
than desired, but reasonable exposure of the reflections that are
present. Use of very long exposures requires special attention to
background; in particular, the 2K CCD has relatively high dark noise,
which may be a problem in such cases. Otherwise, the same considerations
apply as for shorter exposures.
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The approach of taking very
narrow oscillation ranges ("fine phi-slicing") has been tested at CHESS.
In this case, an initial exposure with a wide oscillation range should
be taken to evaluate crystal mosaicity. Selection of exposure time is
done using a narrow-oscillation image. Total rotation range is
determined as usual. When considering the fine phi-slicing approach, the
readout time of the detector becomes very important, as does the
available disk storage for data frames. |
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mount a crystal and take
initial shot(s); |
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check for crystal problems,
good exposure time, good spot separation; |
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index image, check mosaicity
and oscillation range; |
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check potential completeness
of data; |
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set experimental parameters
and take data; |
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process data as soon as you
can - plans are nice but the proof of the pudding is in the eating!
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