Our Main Methods
Depending on the preferred geometry of the proteins studied, we employ various lipid layer-mimetics to find the one with optimal properties for our specific sample: this includes simple detergent micelles, amphipols, Saposin A nanoparticles, nanodiscs, lipid vesicles and lipid nanotubes. Furthermore, we actively investigate approaches to customize and optimize these templates, with the aim of applying them for dynamic structure determination.
The focus of our sample preparation for electron microscopy work is to capture dynamic processes. Thus, we concentrate on timed freezing/vitrification and trapping proteins in functional relevant conformations. In addition, we are also employing liquid phase sample preparation for selection questions.
Transmission images are then either directly evaluated to study the ultra-structure of the sample, or used to reconstruct the underlying three-dimensional structures employing either single particle, helical reconstruction or sub-tomogram averaging methods, depending on the geometry and homogeneity of the sample studied.
Electron Microscopy Equipment
The university of Cologne has made a strategic effort to establish high-resolution cryo-EM in Cologne. While we are in the process of establishing a leading-edge facility associated with the Instiute of Biochemistry, we still have full access to the electron microscopy suite available at the research center caesar. For this, we cooperate with the "Electron Microscopy and Analytics" scientific facility lead by Dr. Stephan Irsen for image acquisition. We have access to direct electron detectors, energy filters and phase-plates allowing for a wide range of samples to be imaged under optimal conditions. To prepare cryo samples, we are relying on either an FEI Vitrobot or on a Leica EM GP. Moreover, we are also custom-building a freeze plunger for specific applications.
Samples are then transferred to the microscope and imaged using a variety of cryo-capable holders. In addition to the cryo-capable holders, we are also using a Protochips Poseidon510 holder to image samples in situ (that is in a liquid phase without the need for freezing and thus stalling of the reaction imaged).