Automated high-throughput crystallization suited for membrane proteins at the ESFRI Instruct Core Centre Frankfurt

The ESFRI (European Strategy Forum on Research Infrastructures) initiated within FP 7 six infrastructure projects, one of them called Instruct (Integrated Structural Biology Infrastructure for Europe) is specialized on structural biology. Instruct selected 2007 the Max-Planck-Institute of Biophysics to setup the core centre Frankfurt, which is devoted to membrane proteins. Our work emphasizes on the structure determination of proteins and protein complexes integrated into the cellular membranes of living organisms. The importance for life of membrane proteins can be deduced from a huge variety of duties, for the communication of the cell with its environment, like signal exchange, as well as energy transduction and specific uptake and/or release of substrates of all kind. Within the Core Centre four areas of protein and crystal handling are installed: Verification of the protein by mass spectrometry, thermal stability tests with a calorimetric approach or a thermofluorassay, fully automated crystallization and analysis of the crystals by X-ray diffraction.A thermofluorassay with small sample quantities has been improved for membrane proteins using the dyes Sypro Orange and CPM. This method allows to quickly enhance the starting conditions for crystallization. Membrane protein crystallization still remains a challenging task. The Rigaku CrystalMation^TM system was setup to fully automate the crystallization process while dealing with sample volumes of 100 nl per experiment. Three different crystallization strategies are applied to offer a broad range of crystallization conditions for membrane proteins. Identification of crystallization hits is simplified by UV detection combined with conventional imaging.

Overview CrystalMation