Th step after the purification and proper characterization of a protein sample, we are ready for setting up crystallization screens. Even if the protein has previously been crystallized, often one has to start with a small screen around the published crystallization conditions. Minor differences in the handling of a protein, in its purification, etc. may affect the crystallization conditions.
Protein crystal structure determination requires crystals. After protein characterization conditions for obtaining well-diffracting single crystals need to be identified (if the protein has not been crystallized before). The number of experimental parameters affecting protein crystallization can be very large. Among these is the type of the buffer and the pH of the solution, the ionic strength, the presence of various salts, the temperature of the experiment and the presence of ligands (co-factors, substrate analogues, inhibitors, etc.). In addition, we need to identify the most suitable type of precipitant, which is one of the most central components in the setup. It should be noted that bound ligands often facilitate protein crystallization, presumably by stabilizing a protein structure, reducing potential flexibility.
The most popular precipitants used in protein crystallography include polyethylene glycols of various lengths, ammonium sulfate, and some alcohols. To find the right combination of parameters, one needs to screen a large number of different conditions. Several hundreds, and often thousands of different conditions are screened before finding the crystallization conditions. Commercial screens, like those from Hampton Research or Molecular Dimensions, are used. However, we have the facilities to design our own screens, if required (usually done during optimization of the conditions found using the initial screen).
The crystallization lab at SARomics is equipped with robotics techniques for the most efficient high-throughput crystallization screens. High precision screening and imaging robotics in protein 3D structure determination help saving time and material. For example, we have the capability to screen 96 different crystallization conditions with as little as 15 micro L of protein sample using a Mosquito liquid handling robot. When the conditions are found they normally need to be optimized further to yield crystals suitable for X-ray crystallography.
In the case of our off-the-shelf FastLane structures, we use available expression constructs with established and verified protocols for expression, purification and crystallization. A combination of FastLane structures with our fast access to the protein crystallography beamlines at MAX lab allow us to minimize the time between the first meeting with a new customer and delivery of the final protein structure.