Page 92 - Macromolecular Crystallography

IN-HOUSE MACROMOLECULAR DATA COLLECTION 81

5.4 Other in-house X-ray sources are loosened and the arcs and sledges set to zero
before commencing. Screw the goniometer onto the
Apart from the rotating anode generators described
goniostatthenmounttheloop-orcapillary-mounted
above, there are two other developments in the
crystal onto the goniometer so that it can be viewed
generation of X-ray radiation in-house. The first is
through the telescope or on a viewing screen.
the development of a high-brilliance, sealed tube
Centring is most easily accomplished by rotating
generator by Ulrich Arndt at the LMB Cambridge
the base plate below the crystal by inserting a thin
and then marketed by Bede Scientific Instruments
Allen key (hex-key inAmerica) into a hole in the base
Ltd. This source consists of an ellipsoidal mirror
plate and turning so as to place a crystal face parallel
with the filament at one focus and the crystal at the
to one of the translation sledges (for a crystal with
other. The source uses very low power, 24 W, and
oblong or squarish faces) with the ϕ (phi)-axis on the
has a very high output (Bloomer and Arndt, 1999;
machine set to zero. Then lock the grub-screw secur-
Arndt et al., 1998a, b). This source will clearly prove
ing the goniometer plate so that it will not rotate.
useful where electrical power is restrictive, such
It is possible to centre a crystal mounted at a ran-
as on the space station; therefore protein diffrac-
dom orientation, but to centre on a crystal face is the
tion data could be collected directly after crystals
easiest way of starting and often will place a crys-
have been grown in order to answer vexing ques-
tallographic zone onto the detector. The translation
tions regarding the improvement of crystal quality
sledge is then adjusted with the goniometer key to
after growth under zero-gravity conditions. The sec-
bring the crystal into the centre of the cross-hairs.
ond development is that of a small synchrotron
Unlock the ϕ drive on the goniostat and move to
for the generation of X-rays for in-house protein
◦
ϕ = 90 and repeat the operation to bring the crys-
crystallographic use. This development has been
tal into the centre of the cross-hairs. Crystal centring
possible by using free electron laser technology to
◦
◦
is then checked at ϕ positions of 180 and 270 and
steer the circulating electron beam in a synchrotron
adjustments made so that when the crystal is gently
with a very small diameter and produce synchrotron
rotated on ϕ there is no side-to-side precession of the
radiation in the 1 Ångstrom range. This system is
crystal relative to the cross-hairs. If there is a small
now sold by Lycean and the first one will shortly
side-to-side precession then very small adjustments
be installed in a protein crystallography laboratory
are made to the translations to correct this at posi-
in the US.
tions 0 and 180 ;90 and 270 . This manoeuvre
◦
◦
◦
◦
takes practice and is very important as at the end
5.5 Setting up for data collection of it a perfectly centred crystal is achieved, which
both stays in the beam for the full course of the data
5.5.1 Centring crystals
collection and is centred. As I said above, a crys-
Centring crystals is an activity that is common to the tal face should be aligned parallel with a translation
start of all data collection, so I shall begin by describ- sledge. However, sometimes clear crystal faces may
ing this and then proceed to discuss the specifics of not be distinct and then the centre of mass of the
data collection on two types of image plate detec- crystal is estimated by eye and placed in the cen-
tor (the Raxis-IV ++ and the MAR345) and one CCD tre of the cross-wires and the translation corrections
◦
◦
◦
◦
detector (the MARCCD) Protocol 5.1 and Protocol performed at ϕ = 0 and 90 and then at 0 and 180 ;
◦
◦
5.2. Centring is a procedure that has to be mastered 90 and 270 , as above, so that when the crystal is
and the ability to do it quickly comes with practice. gently rotated there is no side-to-side precession. If
Some goniometer heads are supplied with arcs and a crystal has been cryocooled with a large amount
translation sledges and some with only translation of liquid around it, this may have the effect of act-
sledges. A very commonly used goniometer head, ing like a lens and one has to be careful to correct
which is of very high quality, is made by the Huber adjustments on the translations accordingly. With
Company and these are supplied with and without crystal mounting in a capillary, an amount of liquid
arcs and in a variety of heights. It is best to make left around the crystal in the capillary can have the
sure the arc and translation securing grub-screws same lensing effect.