Page 93 - Macromolecular Crystallography

82 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 5.1 Data collection using the Raxis IV ++ image plate

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The direct beam position is always determined after a parallelpiped) then collect frames at 0 ,45 , and 90 .If
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change in filament and/or realignment of the mirror system. the crystal appears to have a hexagonal or trigonal habit
This consists of placing a thin sheet of attenuating nickel and the six-fold axis is along the direction of the ϕ rotation
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sheet (provided by the manufacturer) on the goniometer so axis then collect frames at 0 ,30 , and 60 .
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that it is aligned normal to the beam. The main shutter is 3. Once the first frame is recorded, determine the
opened for a very short period of time (the length of time is diffraction limit of the crystal from the image. If this limit
dependent on the flux of the X-ray generator) but when is less than 2.8 Ångstroms then stop the collection of the
read should result in a small intense spot at the centre of remaining two frames and adjust the distance to the
the plate. It is important, if one does not know the value of diffraction limit so as to fill the whole plate with diffraction
the exposure, to err on the side of caution and start with a intensities. If the crystal diffracts further than 2.8 Ångstroms
few tenths of a second and then increase the value rather then stop the collection after the first frame and drive in the
than over-expose the central region of the film emulsion to detector to, say, 2 Ångstroms and record three more images.
excessive radiation. Progressively, increase the value until Continue to change the detector distance and take frames
you are satisfied with the number of counts in this region. till the maximum resolution of the diffraction pattern fills
The direct beam spot should be easily cursorable and then the detector. Care must be taken when driving to high
entered into the software. resolution not to collide the back stop or the cryocooling
ALWAYS check that all the safety shutters are closed equipment with the black paper on the front of the detector.
before opening the safety enclosure and making 4. Process the frames using CrystalClear, which is the
adjustments. X-rays are very dangerous and every safety standard software that comes with the instrument, and
precaution should be taken when using them. A new user determine the space group and crystal lattice parameters.
should under no circumstances start using X-ray equipment (Some crystallographers prefer to export either a single
without experienced guidance. frame or all three to process with other software; personally
I prefer to process and set up for collection using
1. The telescope on the Raxis IV ++ is mounted at 45 to CrystalClear and maybe use other software if I have
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the ϕ axis and the goniometer is locked onto the ϕ axis and problems easily getting a space group determination or
the crystal centred as described under (see Centring crystals good integration of the data.) Check for spot overlap at the
above). chosen distance (if there is overlap then the detector will
2. Crank the detector back to a distance so that have to be moved further out). Also select ϕ so that there
2.8 Ångstrom is collected at the edge. A calibration chart is no spot overlap in the ϕ direction. If there is overlap then
mapping resolution in Ångstroms at the edge of the ϕ must be decreased.
detector with distance should be readily available to the 5. It pays to process the data as it is collected, to observe
operator, either in paper form or on the computer terminal. how far the crystal diffracts in different crystal orientations
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Collect three oscillations of 1 . If the crystal habit looks as and how well it merges in terms of R factor in resolution
if it is either monoclinic, orthorhombic, or tetragonal (i.e. shells and overall cumulative R factor.
the crystal looks like a small ‘shoebox’, a rectilinear

5.6 Image plate detectors and when exposed ‘colour centres’ are raised to an
excited state. Once the plate has been exposed for the
Image plates are made by coating a flexible, matt
requiredperiodoftimetheplateis‘read’byscanning
plastic backing sheet with a thin layer of emul-
across the plate with a finely focused HeNe laser. The
sion (150 µm) consisting of a europium compound
‘colour centres’ then fall in their energy levels and
BaFBr(Eu). They are primarily sold by Fuji and are
emit visible light, which is read by a very sensitive
now available for a wide range of imaging purposes, photomultiplier and the analogue signal converted
ranging from 32 P gel scanning in molecular biology,
into electronic output. For reuse the plate is ‘flashed’
X-ray crystallographic detectors, through to medical
clean by exposing it to light from the erasing lamps.
X-ray imaging. This emulsion is sensitive to X-rays
Effectively, the image plate is like a reusable piece