Page 190 - Macromolecular Crystallography

HIGH-THROUGHPUT DATA COLLECTION AT SYNCHROTRONS 179

crystals (i.e. <50 µm in diameter) make sample Non-X-ray signatures from crystals can also be
positioning akin to threading a needle. For larger used for detection/centring. Ultraviolet fluores-
sized beams, such as those found with home sources cence from the aromatic residues in the polypeptide
or at older synchrotrons, or crystals with greater chain can indicate the position of the crystal within
dimensions, this process can be considerably easier. the mount. Recent work has utilized high inten-
With appropriate crystal lighting and video-based sity UV lasers for fluorescence excitation (Vernede
visualization optics, it is usually possible for an end et al., 2006). Differential transmission and reflection
user to locate the crystal within the nylon loop and of infrared radiation from the crystal, relative to that
position it in the X-ray beam. At present, automa- of the loop and the surrounding cryoprotectant, has
tion of this process does entail some compromise. also been used to detect crystals within nylon loops
With current technology, it is considerably easier to (Snell et al., 2005). The success of this latter tech-
identify the centre of a quasicircular nylon loop than nique at a synchrotron beamline is not yet at the
to recognize a protein crystal, particularly because level achieved in the developmental laboratory. One
individual crystals can vary greatly in both shape of these methods may ultimately become a stan-
and size. Centring based on detection of the loop, dard means for detecting protein crystals directly.
however, will place the crystalline sample in the Currently, however, control of the location of the
X-ray beam only if the diameter of the loop is com- crystal within a commensurate loop prior to cryo-
mensurate with that of the crystal or the beam. This genic preservation in liquid nitrogen, followed by
centring method, therefore, requires upstream con- positioning of the loop, represents the most reli-
trol of how crystals are mounted, ensuring that the able method for rapid, accurate placement of protein
loop and the crystal are matched in size and that the crystals in X-ray beams at synchrotron sources.
crystal is placed at or near the centre of the loop. Illu-
mination of the loop and visualization optics have a 12.3.5 Removal of surface ice
significant impact on automated recognition of the
crystal mount. The lighting strategy must provide It is common for liquid nitrogen frozen protein crys-
sufficient contrast between the loop and the back- tals to acquire a patina of ice on the surface of
ground of the video image and minimize glare from the cryoprotectant. Diffraction of X-rays from even
the surfaces of cryogenically frozen samples. Sub- small ice crystals can mask reflections from the pro-
optimal lighting can obscure the boundaries of the tein crystal. In addition, the presence of excessive
loop and compromise the effectiveness of automated amounts of ice can obscure the true position of the
detection algorithms. nylon loop, thereby resulting in the failure to place
For high-throughput data collection, sample cen- thecrystalintheX-raybeam. Itis, therefore, essential
tring via loop detection is currently the method of to remove ice crystals prior to diffraction analysis.
choice for placing crystals in the X-ray beam. Alter- Traditionally, crystallographers have manually
native methods rely on the direct detection of the removed ice by either pouring a small amount of liq-
crystal itself by monitoring the intensity of either uid nitrogen over the crystal or by gently abrading
X-ray diffraction from the crystal or X-ray fluores- the surface with an implement such as a single paint
cence from an element in the crystal that is not brush bristle. The Stanford Auto-mounting System
present in the cryoprotectant (Pohl et al., 2004). Both provides for removal of a sample from the goniostat
of these approaches have drawbacks. First, some of and rapid ‘washing’ within a bath of liquid nitrogen
the limited X-ray lifetime of the crystal (Section 12.6) to eliminate ice (Cohen et al., 2005).
must be committed to detection instead of data col- SGX-CAT uses a unique system based on a small
lection. Second, the loop itself has to be placed close liquid nitrogen pump for a similar purpose. The
to the X-ray beam prior to crystal detection, followed pump controls delivery of a gentle stream of liquid
by step-wise translation of the crystal through the nitrogen that is directed onto the surface of the sam-
beam, thereby increasing the time required to centre ple after it has been positioned within the gaseous
the sample. cryostream by the sample-changing robot. A phase