Page 71 - Macromolecular Crystallography

60 MACROMOLECULAR CRYS TALLOGRAPHY

improved). The appropriate experimental protocol discussed in the following sections. The first two
will vary with the crystalline sample. methods involve replacement of solvent in/around
the crystal with a synthetic buffer, while the third
4.2.1 Identification of a cryostabilization approach involves replacement of solvent on the
buffer surface of the crystal with various types of hydro-
carbons. The method of cryoprotection will usually
Preparation of a crystal cryostabilization buffer for a
vary with the crystalline sample.
newly prepared macromolecular crystal is an exten-
sion of the efforts to devise a crystal stabilization
buffer; in some cases the buffers may be one and
4.2.2.1 Slow equilibration in cryoprotection buffer
the same. The major objective is to introduce the
Slow equilibration into a cryostabilization buffer is
crystal – in a controlled manner – into solutions con-
carried out by serially transferring the macromolec-
taining synthetic mother liquor supplemented with
ularcrystalintoaprogressivelyhigherconcentration
an antifreeze agent, which include various poly-
of cryosolvent. This procedure is initiated by trans-
hydric alcohols, low molecular weight polyethy-
ferring crystals into synthetic mother liquor supple-
lene glycols, sugars, organic solvents (Garman and
mented with 5% antifreeze agent. Crystal transfers
Schneider, 1997), and salts (Rubinson et al., 2000)
can performed using a fibre loop, as described
(Table 4.1). Making the assumption that the intro-
below. The crystal is then serially transferred into
duction of any non-native component into a crystal
a progressively (e.g. 5% steps) higher concentra-
has the potential to cause damage, the investigator
tion of antifreeze agent until the concentration of
should aim to use the lowest concentration that is
agent determined to be non-damaging is reached.
necessary to cryopreserve the crystal for data collec-
Allow the crystal to equilibrate in the cryostabiliz-
tion (Mitchell and Garman, 1994). The nature and
ing buffer for 15 min to several hours or even over
concentration of the appropriate cryosolvent will
night; the equilibration time will vary with the crys-
vary with the crystalline sample.
tal. The time required for diffusion into a crystal is
dependent on many factors (size of small molecule,
4.2.2 Transfer of macromolecular crystals into temperature, size of crystal, nature of channels in the
cryostabilization buffer
crystal, etc.). The little data that exists suggests that
The three common methods in use for perform- this time could be on the order of minutes to hours
ing crystal cryoprotection prior to shock-cooling are (Wyckoff et al., 1967).
Variations to the slow-equilibration method
Table 4.1 Antifreeze agents used in the shock cooling of include cross-linking of crystals prior to transfer
macromolecular crystals into cryobuffers (Lusty, 1999), transfer of crystals
into cryobuffer by dialysis, or the introduction of
Agent Concentration range (%)
the cryobuffer during crystallogenesis.
Glycerol* 10–40
Ethylene glycol 10–40
Propylene glycol 10–40 4.2.2.2 Rapid passage through cryoprotection buffer
2,4 Methyl pentane diol 10–40 Rapid passage, also known as the ‘quick-dip’
Sucrose up to 30 method, involves mounting a crystalline sam-
Alcohols (methanol, ethanol, up to 10 ple in a fibre loop and rapidly (1–5 s) drag-
isopropanol) ging the sample through a cryostabilization buffer.
Low molecular weight up to 25
This method is especially useful for crystalline
polyethylene glycols
samples that are damaged by prolonged exposure
Mineral oil 100
to antifreeze-containing buffers. The simplicity and
Salts (Lithium, sodium, and varies
speed offered by the ‘quick-dip’ method makes
magnesium salts)
it attractive when rapidly preparing shock-cooled
Mixtures of the above varies
samples.