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Efficient collection of
oscillation data:
planning,
pitfalls, and prospects
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| Most macromolecular
crystallographers coming to CHESS collect diffraction data using
the oscillation method. This is a time-tested and well
understood technique, but several factors must be considered to
collect good oscillation data in the most efficient way
possible, so as to make best use of one's limited synchrotron
time. Here we consider some of these factors, and mention how
new software at CHESS will help users plan more efficient data
collection. |
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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 2 |
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From the image alone several things can be checked: |
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Singleness of
crystal |
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
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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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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 |
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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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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Contents |
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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 |
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 |
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 |
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 |
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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Contents
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