Page 64 - Macromolecular Crystallography

AU TOMATION OF CRYS TALLIZATION TECHNIQUES 53

to ‘back off’ to conditions of growth (Fig. 3.3). After a given time (before crystals are visible), the
Dilution can be achieved using all crystallization coverslips are transferred over reservoirs containing
methods. Protocols for ‘backing off’ experiments in lower precipitant concentrations that would nor-
the most common techniques, namely microbatch mally yield clear drops (Fig. 3.4). As in dilution,
and vapour diffusion, are given in Protocol 3.5. the time of transfer is selected by reference to the
time which it took to see the first crystals in the
initial screens. The transfer lasts 1–2 seconds. This
3.5.3 Determination of the timing of dilution technique has produced significant improvement in
crystal order of a number of proteins (e.g. Saridakis
Dilution must be performed before crystals are vis-
and Chayen, 2000, 2003; Krengel et al., 2006).
ible. The time of dilution is selected by reference
to the time which it took to see the first crystals in
the initial screens (Saridakis et al., 1994). For exam- 3.5.5 Control of evaporation in microbatch
ple if crystals appeared within 24 hours, nucleation
would have occurred anytime between setting up An alternative to dilution in microbatch is to app-
the experiments to several hours before the crystals roach the optimization strategy from the opposite
appear. Hence the dilution should be done at inter-
vals of 1–2 hours after set up. If crystals appear after Robotic arm
4 days, dilution should be performed at intervals of X hours
8–12 hours. Trials that are diluted too soon will pro-
duce clear drops while those that are too late will
yield low-quality crystals.
Crystallization
drop
3.5.4 ‘Backing off’ experiments in hanging drops
Dilution experiments involve revisiting the crystal-
lizationdropswhichmaycausedisruptiontothetrial. Reservoir Reservoir with
An alternative way of backing off without touching lower precipitant
concentration
the trial drop can be achieved in hanging drops in
the following way : the coverslips holding the drops Figure 3.4 Transfer of a hanging drop from nucleation to optimal
are incubated for some time over reservoir solutions growth conditions. Modified from Chayen and Saridakis (2002), Acta
thatnormallygivemanysmallcrystals(Protocol 3.6). Cryst. D 58, 921.

Protocol 3.7 Induction and subsequent arrest of nucleation in microbatch
Equipment and reagents 2. Allow to incubate for a given time.
Microbatch plates 3. Top up the oil (at different times for the different trays)
Paraffin oil so that it fills the dish (Fig. 3.5b).
Protein solutions 4. Continue to incubate as standard microbatch trials.
5. Observe daily.
Precipitant solutions
Pipette or robot
A robot will first dispense the trials under the lower volume
Crystallization reagents
of oil. A robotic arm is programmed to add oil at various
Method time intervals after setting up the trials.
1. Set up several crystallization plates containing
microbatch trials under a layer of paraffin oil so that
the oil just covers the trials (Fig. 3.5a).