Protein crystallization represents a major bottleneck in the determination of high resolution three dimensional protein structures. For most initial crystallization conditions, the resultant protein crystals are of insufficient quality to permit the collection of complete diffraction, at high resolution and low mosaicity.  While some initial crystals come close to the final crystallization condition, many suffer from one or more of these common problems:
 
  • insufficient crystal size
  • intergrown crystals or small crystal clusters
  • poor reproducbility
  • high internal disorder
  • poor resolution
  • anisotropic diffraction
  • high mosaicity
  • incompatibility with cryoprotectants
  • occluded ligand binding sites for soaking and/or cocrystallization trials

 

 

 

The identification of an initial crystallization condition in any format, including vapor diffusion, is an excellent prognostic indicator for the possibly of success of a crystallization project, no matter the quality of the initial hit. Traditionally, the most common approach to addressing the aforementioned problems centers on continued screening of optimization or de novo conditions in the same crystallization format.  The Crystal Former, however, affords significant advantages as a rescue method for projects stuck at the initial crystallization stages.

 

How can a Crystal Former rescue my vapor diffusion hit?

The establishment of continuous gradients between protein and components of the crystallization condition permits the exploration of both initial and optimized crystallization conditions wiithin a single channel. Thus, even for the same condition as found in vapor diffusion, the Crystal Former is able to comprehensively sample local crystallization space, thus improving crystal reproducibility and increasing the likelihood that regions favoring the growth of single, diffraction-quality crystals will be identified.  

Beyond optimization of the vapor diffusion condition, the ability of the Crystal Formers to efficiently explore more of crystallization space also increases the likelihood that an independent crystallization condition will be identified.  Changes in crystal packing can lead to improved internal order and the potential for more accessible binding sites.

Finally, the favorable mixing kinetics at the microscale, exploited in the Crystal Former microchannel, leads to the growth of superior protein crystals with reduced protein requirements.

 

What does this really mean for my research?

High priority projects can be expedited through the use of limited protein sample and a small number of Crystal Formers to save significant time and costs in moving from initial hit to final protein structure.  For drug-discovery targets, the identification of additional crystal polymorphs will provide more rapid determination of protein-ligand complex structures, furthering the overall drug development pipeline.

 

What should be my first step?

We strongly believe in rescreening any target in the SBS High Throughput Crystal Former using a non-redundant crystallization screen that has been optimized for diffusive mixing.  This first step will ensure that you are working with the best possible starting condition for your target, rather than focusing in on the only hit from vapor diffusion. Since capillary-based methods increase the number of unique crystallization conditions by at least 2-fold (and in some cases 10-fold or better), this is a crucial step.  In most cases, a condition similar, if not identical, to the initial vapor diffusion condition will be returned in this screening step with the added benefit of optimization of the condition within a single Crystal Former channel.