Page 74 - Macromolecular Crystallography

FIRST ANALYSIS OF MACROMOLECULAR CRYS TALS 63

of the cryovials is slightly above the level of the liquid motions should be fluid and smooth and might require
nitrogen. Liquid propane should be allowed to thaw in the some practice. Shock cooling by this method can be
cryovials and achieve temperature equilibration with the performed with crystalline samples equilibrated at any
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liquid nitrogen bath (1–3 min). temperature (e.g. 25 C, 4 C, etc.).
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3. A cryoprotected and loop-mounted crystal is 4. Propane in the cryovial should be permitted to solidify
shock-cooled by rapid plunging it into a cryovial containing (5–10 min). The loop-mounted crystal will thus become
liquid propane. The time that the mounted crystal spends entombed in solid propane. The cryovial containing the
exposed to air should be kept to a minimum to avoid drying shock-cooled sample may be transferred to a cryocane for
of the sample. Failure to plunge the loop-mounted sample storage in a long-term storage Dewar vessel. Shock-cooled
into propane rapidly enough will damage the sample due to crystals prepared in this way may be stored in a storage
the formation of crystalline ice. During this procedure, hand Dewar indefinitely.

Protocol 4.2 Shock-cooling into liquid nitrogen
Materials

Items from Protocol 4.1, with the exception of propane of the sample. Shock cooling of crystalline samples into
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Crystal wand (available from Hampton Research, Inc.) liquid nitrogen (t =−180 C) can be performed with
Cryotongs (available from Hampton Research, Inc.) crystalline samples that are equilibrated at any temperature
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Vial clamp (available from Hampton Research, Inc.) (e.g. 25 C, 4 C, etc.).
2. While in the liquid nitrogen bath, the sample (held by
Procedure (Fig. 4.3) the magnetic wand) is manoeuvred into a cryovial, which is
1. A loop-mounted crystal is attached to the magnetic held submerged in the liquid with a vial clamp.
crystal wand, which is used to rapidly plunge the crystal into 3. Once secured in a cryovial, the processed samples are
liquid nitrogen. The time that the mounted crystal spends placed on cryocanes, which are placed into a liquid nitrogen
exposed to air should be kept to a minimum to avoid drying Dewar vessel for storage.

Protocol 4.3 Shock-cooling into gaseous nitrogen

Materials cold-streams include hardware to enable the temporary
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Gaseous nitrogen stream (t =−180 ) diversion of the stream from the crystal.
X-ray diffraction camera 3. With the cold nitrogen stream diverted, the
loop-mounted crystal is positioned on the goniometer head.
Procedure (Fig. 4.4) 4. Rapid cooling of the sample is achieved by quickly
1. The X-ray camera is prepared to accept a loop-mounted removing the obstruction, which re-establishes the original
sample. The X-ray camera should be properly aligned with flow of the stream. The time that the mounted crystal
the X-ray beam and the cold nitrogen cold stream should be spends exposed to air should be kept to a minimum to
aimed at the eucentric point of the X-ray camera. avoid drying of the sample.
2. The gaseous nitrogen stream (which is aimed at the 5. Samples prepared in this way are ready for X-ray
presumptive location of the cooled crystal) is temporarily diffraction or can be recovered (see below) for storage and
diverted with an obstruction such as an index card. Some later use.