Challenging crystallization obstacles at the ISPC

Shelly Hamer- Rogotner, Joel L. Sussman and Orly Dym

The Israel Structural Proteomics Center

Weizmann Institute of Science, Rehovot 76100 ISRAEL

I’m working as a lab technician at the crystallization unit of the Israel Structural Proteomics Center (ISPC http://www.weizmann.ac.il/ISPC)) at the Weizmann institute.  In the line of my work I’m operating and maintaining our state-of-the-art crystallization and visualization robots that increase the efficiency and accuracy of the protein crystallization experiments. In the last seven years at the ISPC, I have setup thousands of crystallization screening experiments on few hundreds of proteins. This was followed by several of sophisticated optimization experiments, which I design in attempts to yield a single crystal suitable for X-ray analysis. I have also implemented a number of seeding strategies for crystallizing proteins. An interesting example was crystallizing the Conk2 and Conk3 proteins. Cones are small polypeptides with disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine-cysteine disulfides. Conk2 and Conk3 proteins are potassium-channel pore-blocking toxin. These proteins have α helix at their C-terminus, which is crucial for channel binding and they share 53% identity. Initially I managed to crystallize the Conk2 (Fig. 1), and the structure was solved 1.46 Å. However, crystallizing the Conk3 protein was very challenging and in fact yielded no crystals from experiments using different crystallization methods at different temperatures. As last resort I used the Conk2 crystals as seed to preform micro seeding and obtained Conk3 crystals (with 53% identity to Conk2)  (Fig. 2) for which the structure was solved to 2.4 Å.

Fig 1: Conk2 crystals	Fig 2: Conk3 crystals obtained by Micro seeding of Conk2 crystals

Another challenging crystallization experiment was on the synaptic enzyme, acetylcholinesterase (AChE), which is an important target for drug design. AChE inhibitors are employed in the treatment of myasthenia gravis and other neuromuscular diseases; the first generation of drugs for the management of Alzheimer’s disease are also AChE inhibitors. Furthermore, AChE is the principal target of nerve agents and of many insecticides. It took me several years to get single crystals suitable for X-ray data collection of the human AChE (hAChE) in the presence of adenosine 3',5'-monophosphate (AMP). Initially I got crystals from the crystallization screens which did not diffract (Fig. 3). Additive screens yielded nice looking crystals however still did not diffract (Fig. 4). Optimization on the original crystallization conditions as well as on the additive yielded many crystals for which the structure was solved to 2.75 Å (Fig.5).

Fig. 3: hAChE with AMP	Fig. 4: hAChE with AMP and Taurine as additive.	Fig. 5: hAChE with AMP in the presence of Taurine and optimization of the original condition.

In my professional carrier I crystallized hundreds of proteins and protein complexes, some of these proteins are related to human disease, and others including engineered non-natural enzymes and non-natural protein complexes. Some have contributed to the development of drugs.