Page 77 - Macromolecular Crystallography

66 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 4.4 Assembly of samples prepared with propane

Materials around the crystal. The slow thawing properties of propane
Aligned X-ray camera provide a favourable time window to transfer the crystal to
Cold gaseous nitrogen stream (t =−180 ) aimed at the nitrogen stream without exposing the crystal to ambient
◦
rotation centre of the goniometer temperatures. Liquid propane should always be handled
with appropriate hand and eye protection. Prolonged skin
Curved forceps
contact with liquid propane at any temperature can cause
Gloves or finger cots
severe burns due to evaporative freezing (Hicks et al., 1979).
Vial clamp
3. While the propane immediately around the crystal is
Low-form Dewar flask
still solid, a pair of curved forceps is used to lift the pin
Cold crystal wand (containing the loop-mounted crystal) out of the cryovial.
Cold cryotongs The mounting pin is placed on the magnetic mount that is
Face shield held on the goniometer head. The solid propane that
Lint-free tissue remains around the crystal will slowly thaw under the cold
Goniometer head with magnetic mount stream, efficiently transferring the crystal from one cryogen
(propane) to another (gaseous nitrogen). Damage to the
Procedure (Fig. 4.5) sample will probably result if propane around the crystal is
1. The edge of the cold-stream should be positioned permitted to thaw while the sample is outside the
approximately 1–2 cm from the presumptive position of the cold-stream.
crystal. This is a much larger distance than that used during 4. As the propane continues to thaw, the cold-stream
recording of data and is meant to provide an ample work should be brought closer to the sample. Optimally, the tip
space. of the nozzle should be brought as close as possible to the
2. A cryovial is selected and placed on a flat surface at sample without casting a shadow on the X-ray detector.
ambient temperatures. This allows the external most layer of 5. Any liquid propane that remains on the sample should
solid propane to thaw (c. 10 s) and will result in release of be wicked away from around the crystal with a lint-free
the loop/mounting pin from the cryovial. At this point, a tissue. Thawed liquid propane should be collected and
substantial amount of propane will remain in solid form allowed to safely evaporate in a chemical fume hood.

Protocol 4.5 Assembly of samples prepared with liquid nitrogen

Materials 3. The mounting pin (held by the magnetic crystal wand) is
Crystal transfer tongs manoeuvred into the head of the cryotong. Encased within
Curved nose clamp the head of the cryotong, the loop-mounted crystal will
Shallow Dewar flask remain at cryogenic temperatures for up to 20 s.
4. With the cryotongs in their locked position, the crystal is
Crystal wand
lifted out of the liquid nitrogen bath and positioned above
Insulated gloves
the magnetic mount, which is held on the goniometer head.
Face shield
The time that the head of the cryotongs spends outside of a
Procedure (Fig. 4.5) cryogen (liquid or gaseous nitrogen) should be kept to a
1. A vial containing a shock-cooled crystal is placed in a minimum.
bath of liquid nitrogen, which is housed in a shallow Dewar 5. Transfer of the crystal to the cold nitrogen stream is
flask. achieved by quickly unlocking the cryotongs.
2. The pin containing the loop-mounted crystal is removed
from the cryovial and captured with the magnetic crystal Note: Hand and eye protection should always be used when
wand. handling liquid nitrogen.