RESUMEN
Many instrumentation developments in crystallization have concentrated on massive parallelization assays and reduction of sample volume per experiment to find initial crystallization conditions. Yet improving the size and diffraction quality of the crystals for diffraction studies often requires decoupling of crystal nucleation and growth. This in turn requires the control of variables such as precipitant and protein concentration, equilibration rate, and temperature, which are all difficult parameters to control in the existing setups. The success of the temperature-controlled batch method, originally developed to grow very large crystals for neutron crystallography, demonstrated that the rational optimization of crystal growth has potential in structural biology. A temperature-controlled dialysis button has been developed for our previous device, and a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature-precipitant concentration phase diagrams has been constructed. The presented approach differs from the current paradigm, since it involves serial instead of parallel experiments, exploring multiple crystallization conditions with the same protein sample. The sample is not consumed in the experiment and the conditions can be changed in a reversible fashion, using dialysis with a flowing precipitant reservoir as well as precise temperature control. The control software allows visualization of the crystals, as well as control of the temperature and composition of the crystallization solution. The rational crystallization optimization strategies presented here allow tailoring of crystal size, morphology and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination.
RESUMEN
In protein crystallography experiments, only two critical steps remain manual: the transfer of crystals from their original crystallization drop into the cryoprotection solution followed by flash-cooling. These steps are risky and tedious, requiring a high degree of manual dexterity. These limiting steps are a real bottleneck to high-throughput crystallography and limit the remote use of protein crystallography core facilities. To eliminate this limit, the Robotic Equipment for Automated Crystal Harvesting (REACH) was developed. This robotized system, equipped with a two-finger micro-gripping device, allows crystal harvesting, cryoprotection and flash-cooling. Using this setup, harvesting experiments were performed on several crystals, followed by direct data collection using the same robot arm as a goniometer. Analysis of the diffraction data demonstrates that REACH is highly reliable and efficient and does not alter crystallographic data. This new instrument fills the gap in the high-throughput crystallographic pipeline.
