Page 63 - Macromolecular Crystallography

52 MACROMOLECULAR CRYS TALLOGRAPHY

but of course can be done far quicker if performed fragile crystals and, other than streaking, seeding
using robots. manoeuvres are not very simple to perform.
An alternative means of conducting trials with a
similar outcome to seeding, but without the need to
3.5.2 Separation of nucleation and growth
handle crystals, is by dilution. An additional benefit
The most commonly used technique for separation of dilution methods is that they are more amenable
of nucleation and growth is seeding (Stura, 1999; to automation compared with seeding techniques
Bergfors, 2003; and references therein). Although (Chayen, 2005). The aim of dilution is to start the
very successful, seeding often involves handling of trial at nucleation conditions and after a given time

Protocol 3.5 Dilution in microbatch and vapour diffusion
Equipment and reagents 2. Dilute the trials at given times after set up by adding a
Microbatch plates volume of buffer or of protein in buffer at a volume which is
Paraffin oil 5–10% of the total drop volume.
Pipette or robot
In microbatch, the crystallization drops are diluted directly.
Plates for hanging or sitting drops
In the case of vapour diffusion, either the drops themselves
Coverslips
or the reservoirs can be diluted.
Sealing tape If performing manually, use a pipette of your choice. If
Protein solution performed by a robot, the robotic system is programmed to
Buffer revisit the drops and/or the reservoirs at given times in
order to add the diluent (Chayen, 2006).
Method
1. Set up trials under conditions that would give you the
low-quality crystals.

Protocol 3.6 ‘Backing off’ in hanging drops

Equipment and reagents 3. Set up 6–10 trials under conditions that would give you
Linbro type plates the low quality crystals.
Any oil of viscosity of ∼5 4. At a given time after set up (based on when the first
Pipettes crystals were seen as explained in Section 3.5.3) transfer
(either manually or by a robot) one of the cover slips onto a
Coverslips
reservoir containing the lower precipitant concentration
Pasteur pipette with rubber squeezer
(Figs 3.3 and 3.4).
Protein solution
5. After a further time interval transfer a second cover slip,
Precipitating agents
then the third, fourth, etc. (Figs 3.3 and 3.4).
6. Leave one or two drops without transferring them and
Method
1. Prepare 6–10 reservoirs with a solutions containing one or two drops under the low precipitant concentration to
precipitant concentration that would result in producing a act as controls.
clear drop if crystallization drops were set up and left to 7. Incubate and wait at least 1 week. The time for the
incubate under these conditions. Determine these formation of crystals will be longer in the transferred drop
concentrations from the region in the phase diagram that is compared to the control experiment that had not been
just under the supersolubility curve (Fig. 3.3). transferred, however the crystals at some of the transfer
2. Grease the rim of the plate with oil using a Pasteur times are likely to be fewer and better ordered.
pipette and cover the reservoirs with cover slips.