Page 94 - Macromolecular Crystallography

IN-HOUSE MACROMOLECULAR DATA COLLECTION 83

Protocol 5.2 Data collection using the MAR345 and the MARCCD using the MARDTB

1. Move the detector back to ≥200 mm to give clearance separated at this distance. If not, drive the detector
and to avoid damaging the face of the detector. further out and record frames until they are.
2. Move the back-stop out of the way. This is done using 9. From the oscillation and the newly derived distance,
the ‘remote control’ unit next to the instrument. calculate the orientation matrix using the strategy
3. Check that ϕ (PHI) on the drum of the goniostat is set programme within the MAR or by another software
◦
to 0 . Check that χ (CHI) is set to zero. (Some cryocooling package. Determine the start ϕ and the oscillation range
◦
users like to set χ = 60 or 70 ; the remote control unit in order to collect the highest percentage of the total
allows toggling CHI between 0 and 60 .) obtainable data in the shortest time. Confirm that along ϕ
◦
4. (a) Mount the ‘crystal and the goniometer’ onto the there are no overlaps, otherwise ϕ has to be decreased.
goniostat. Centre the crystal and align it to the centre of the 10. Optimize the beam using the beam optimization
‘cross-wires’ using the small viewing screen (see Centring features on the MARDTB.
crystals above). 11. Start collecting the data. Try and process the data ‘on
(An option on the MARDTB is to have motorized x, y the fly’ during collection and perform data reduction on it in
translations built into the goniometer; z-translation along ϕ order to see how well it merges. Also inspect the frames
is always available. When x, y motors are present an IUCr, closely during data collection, looking for crystal pathologies
Huber goniometer is not used, but the crystal is mounted such as spot splitting and changes in the resolution of
directly onto the ϕ axis with a magnetic cap.) crystal diffraction at different crystal orientations.
(b) Replace the back-stop.
5. Move the detector to 80 nm for the MAR345 or
35 mm for the MARCCD165 (corresponding to a resolution Useful points to note
of 1.4 Ångstroms). Unless the crystal being studied is a DNA For the MAR345 a distance of 100 mm (50 mm for
crystal or an exceptionally well-diffracting protein then the the MARCCD165) corresponds to a resolution of
final observed is likely to be less than this. 1.54 Ångstrom when using Copper Kα radiation.
6. Check the crystal diffraction by setting up to take a Distance to resolution conversions can be calculated
◦
0.5 oscillation (for around 2 min) by specifying the using simple trigonometry by using the expressions
required parameters in the file, namely crystal name, (for zero detector swing):
distance, and oscillation range. If the diffraction is weak,
repeat the exposure with a longer time. Resolution = ∗ −1
∗
7. Index the image and look for tell tale pathologies in 1.54/(2 sin[0.5 tan (172.5/x)]) for the MAR345
the image, such as spot splitting and high spot mosaicity. Resolution = ∗ −1
∗
If the crystal looks ok, proceed. 1.54/(2 sin[0.5 tan (82.5/x)]) for the MARCCD
8. Determine the resolution limit of the crystal from this where the wavelength is assumed to be that for Copper Kα,
image and drive the detector to the distance such that the namely 1.54(178) Ångstroms and x is the crystal to detector
edge of the detector corresponds to this diffraction limit, i.e. distance.
the diffraction image should fill the detector once recorded If no image appears initially then this may be because
at this distance. Confirm that the diffraction spots are well the erase lamp has blown; this may happen especially at
synchrotrons, where they see much higher use.

of photographic film without the need for chem- 5.6.1 Raxis-IV ++
ical processing and time-consuming scanning, as
Thedetectorconsistsof30 cmsquareplatesmounted
was the case when film was used to record macro-
onto a flexible belt. Once exposed the plate is driven
molecular X-ray data. Three image plate systems are
round to the back of the detector and sucked by
now marketed by Rigaku MSC: the Raxis HR (with
vacuum onto the inside of a metal hemicylinder. The
350-mm plates for high resolution work); the Raxis-
plate is read out by a revolving mirror which spins
IV ++ ; and the Raxis-HTC (with three 300-mm image
the length of the axis of the cylinder and, while pro-
plates and a very fast readout by the use of a dual
gressing incrementally, illuminates the plate with
read head).