Crystallization, Structure Determination and Structure Modeling Unit
The Crystallization, Structure Determination and Structure Modeling Facility at the Structural Proteomics Unit (SPU) within the Life Science Core Facility employs multiple parallel approaches to determine and analyze the 3D structures of proteins and protein complexes. This process requires the seamless integration of expert knowledge with state-of-the-art robotics available at the facility.
Protein crystallization aims to produce well-ordered protein mono-crystals large enough to diffract X-ray beams. This trial-and-error process involves screening various precipitants, testing different crystallization methods, utilizing various robots, and operating at two temperatures, 4ºC and 19ºC. Once suitable crystals are obtained, X-ray data are collected using either the in-house revolutionary X-ray Liquid Metal Jet (LMJ) diffraction system or at a synchrotron facility. The LMJ system can collect high-resolution X-ray diffraction data from a single protein crystal at cryogenic temperatures. A unique feature of the LMJ system is its capacity to collect X-ray data directly from crystallization plates at room temperature (RT). This data is then used to solve the protein's 3D structure. Detailed comparative sequence-based structure analyses of the obtained protein structure and related proteins is performed, gaining insights into their mode of action, specificity, and biological relevance.
Crystallization and Structural Determination Unit Activities
- Preliminary Bioinformatics Research: Providing assistance in planning the choice of constructs.
- Crystallization Screeinig and Optimization: Utilizing methods such as sitting and hanging drop by vapor diffusion, microbatch under oil, and Lipidic Cubic Phase (LCP) for crystallizing soluble and membrane proteins. The Mosquito and LCP Mosquito crystallization robots are used for these processes.
- Automated Iimaging for Protein Crystallization Experiments: All crystallization experiments are stored, visualized and imaged automatically using the Formulatrix robots one maintained at 4ºC, and one at 19ºC.
- X-ray Data Collection: Utilizing the Liquid-Metal-Jet (LMJ) X-ray Diffraction System combined with a pixel detector. The LMJ system is capable of conventional data collection using a single protein crystal at cryogenic temperature and has a novel feature for in-situ diffraction data collection at room temperature (RT) from multiple microcrystals directly from crystallization plates. If necessary, X-ray diffraction data can also be collected at the European Synchrotron Radiation Facility (ESRF) in Grenoble.
- 3D Model Building, Structure Refinement, and Validation: Following successful X-ray data collection, a 3D model of the protein is built, refined, and validated.
- Structure Determination Utilizing Cryo-EM Data: Cryo-EM data collected by the Electron Microscopy (EM) unit is used to produce a 3D model of the protein, which is then refined and validated.
- In Silico Predictions: Predicting structures of proteins, protein complexes, proteins and peptides, and proteins with small-molecule ligands. Various computational modeling methods are used, including AlphaFold3, AlphaFold2, RoseTTAFold, RaptorX, Modeller, Swiss-model, and others. The quality and accuracy of the predicted models are assessed to address the investigator's questions and correlate the structural data with biological findings.
- Protein Structure Analysis: Involves sequence-based structure comparison with related proteins and correlating the structure obtained by X-ray Crystallography, Cryo-EM, or in silico modeling, with biochemical data to gain insight into its mode of action, specificity, and biological relevance.