The Role of Oil in Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 137

Filtration of a crystallization trial through a 0.1 micrometer filter can pre-
vent nucleation under conditions previously considered standard (Chayen
et al ., 1993; Hirschler et al ., 1995). Providing the trial remains under oil,
nucleants can be inserted in a controlled manner since one is ensured that
the oil prevents any other contaminant entering the trial, other than that
which the experimenter wishes to add. Experiments have been performed
in which the nucleation, and consequently the number and size of several
protein crystals, was determined at will by the addition of different quan-
tities of a nucleant to filtered trials containing these proteins (Blow et al .,
1994; Chayen et al ., 1993, 2006). The cleanliness of such trials produces
highly reproducible results.
Effect of surface contact
It has been reported that heterogeneous nucleation which is often detri-
mental to the production of diffraction quality crystals can be induced by
the contact of a crystallization trial with the walls of its supporting vessel
(Blow et al ., 1994; Yonath et al ., 1982). The nucleation properties of such
solid surfaces can be manifested even after filtration. A series of experi-
ments shown in Fig. 4 demonstrates how the application of oil can deter-
mine the contact area between the trial and its supporting vessel, thus
enabling the experimenter to monitor the nucleation and reduce or
increase its level at will. The figure illustrates three situations: (a) repre-
sents a drop which has been dispensed onto the floor of a vial and then
covered by a layer of oil; the drop spreads out and flattens over the floor
of the container. (b) shows a drop dispensed into oil as performed in the
normal microbatch procedure (shown in Fig. 1); the drop forms a round
shape with just a small part of it touching the floor. (c) represents a situa-
tion of “containerless crystallization” in which a crystallization drop is
suspended between two oils of different densities as described by Chayen
(1996) and by Lorber (1996). The oils are not miscible and the drop floats
at the interface, thereby not touching the container walls.
The number of crystals produced by procedures (b) and (c) is sig-
nificantly reduced and their size is much larger compared with those