To aid in structure-based drug design targeting neglected tropical diseases, CDIPD has set up a shared crystallography core facility in the second floor of the Pharmaceutical Sciences Building at the University of California San Diego. This facility includes state-of-the-art instruments required to express and purify protein targets, screen and optimize crystallization conditions, monitor crystal growth in the crystallization drops, and finally, harvest and test crystals for X-ray diffraction.

Our equipment includes two Innova 44R and two Innova 42R refrigerated stackable incubator shakers [1] to grow cells overexpressing proteins. Fluid processor Microfluidics M-110P [2] allows quick, efficient and consistent cell lysis for protein recovery. Proteins are purified to homogeniety using fast protein liquid chromatography (FPLC). The laboratory also houses AKTA pure chromatography system [3] configured to purify from microgram to tens of grams of target product. The system is equipped with the multi-angle static light scattering detector, miniDAWN TREOS (Wyatt Technology) [4], which allows absolute characterization of the molar mass and size of the macromolecules.

Purified macromolecules are subject to crystallization, alone or in complex with their inhibitors. Here our "robot-insects" come into play. Mosquito Crystal and Mosquito LCP robots [5]  rapidly determine optimal conditions for protein crystallization. Mosquito uses disposable positive displacement tips to dispense extremely low volumes. Thus, small amounts of precious, highly purified material is required for screening of hundrids of crystallization conditions. Dragonfly [6] is another valuable addition to screen optimization workflow as it can dispense any volume into any well to quickly configure custom 96-well crystallization plates. It is accessorized with the MX one mixer [7] which replaced conventional plate shakers. The MX one keeps the plate stationary, allowing for extremely fast mixing of even the most viscous solutions using high-speed oscillation of a disposable pin array.

Finally, the Rock Imager 2 (Formulatrix Inc.) [8] is used for crystal detection and visualization in the automated mode, both in UV and visible light. The Imager is complemented by the stereo polarizing microscope Leica M165 [9] with LCD camera for viewing and documenting crystallization experiments and for crystal harvesting.

Larissa Podust, Tracy Handel and Fang Sheng were responsible for the design and instrumentation of the facility. Fang Sheng, a manager of the Crystal Lab, provides training to the new users.

[1] Innova 44R and Innova 42R (Eppendorf) - refrigerated stackable incubator shakers

[2] Microfluidics M-110P (Microfluidics) - fluid processor for cell disruption without compressed air or hydraulic cooling water

[3] AKTA pure (GE Healthcare Life Sciences) - chromatography system operated by the UNICORN software

[4] MiniDAWN TREOS (Wyatt Technology) - Multi-Angle static Light Scattering (MALS) detector

[5] Mosquito Crystal and Mosquito LCP (TTP LabTech) - liquid dispensing robots for dispensing extremely low volumes with high accuracy

[6] Dragonfly (TTP LabTech) - liquid dispensing robot for preparation of complex custom optimization screens

[7] MX one (TTP LabTech) - mixes any liquid in small wells with no risk of spillage

[8] Rock Imager 2 (Formulatrix) - automated HTS crystal imaging system

[9] Leica M165 (Leica Microsystems) - stereo microscope