Cryo Electron Tomography
Phone +49 - 89 - 8578 2642
Dr. Jürgen M. Plitzko
Phone:+49 - 89 - 8578 2645
The research program of the department is focussed on the cellular pathways of protein folding and degradation and their crossroads. In identifying and characterizing the players involved in those pathways, we use approaches ranging from genomics and proteomics to biochemistry and biophysics. Electron microscopy has a particularly important role in the repertoire of techniques we use for probing the structure and functions of the molecules we are interested in and major activities aim at developing this technique further.
The Department had, and continues to have, a pioneering role in the development of cryo-electron tomography which is widely seen now as a method of unique potential for visualizing macromolecular complexes in their functional cellular context. It holds great promise for bridging the gap between molecular and cellular structural biology.
The cryo-EM methods for obtaining 3D data can be summarized as tomographic or at least quasi-tomographic. They all rely on the fact that the parallel projection of a 3D object corresponds to a slice in the 3D-Fourier space of the object. In order to obtain the 3D information, different slices of the Fourier space, i.e. projections in real space, have to be gathered to sample the entire information. Different orientations of the sample can be realized by changing the orientation of the specimen. For this purpose the specimen has to be rotated, as it is the case in cryo-electron tomography, or identical copies of a specimen that occur in different orientations can be reconstructed to a 3D model as in 'single particle' analysis.
The visualization and interpretation of tomograms at the ultrastructural level requires decomposition of a tomogram into its structural components, e.g., the segmentation of intracellular membranes or the assignment of organelles. Continuous structures are relatively easy to recognize and delineate, in spite of the low signal-to-noise ratio present in cryo-tomograms. For example, visualizing the organization of the cytoskeleton in Dictyostelium discoideum was possible at the level of individual filaments, without the need for extensive post-processing (Medalia et al., Science 2002, 298).
Although averaging can obviously not be applied to tomograms of unique structures such as individual cells or organelles, such tomograms may nevertheless contain multiple copies of components such as macromolecular machines like the nuclear pore complex, ribosomes, chaperones, or proteases (Beck et al. Science 2004, 306; Brandt et al. Cell 2009, 136). Averaging of cryo-ET data offers some real advantages that are worth the additional efforts, at least in some cases. The most important reason is the ability to image non-purified samples.
Cryo-ET is currently the only technique that can be used for quaternary structure determination of fragile or even transient complexes. The degree of alteration that biological macromolecules undergo under physiological conditions is largely undetermined since there is no existing imaging technique that could resolve them in the context of a cell. Apart from this, averaging of subtomograms also offers one principal advantage compared to averaging from projections: It is fundamentally easier to determine the orientations of an individual copy from 3D data than from single 2D projections.
Upper row: first electron tomographic investigation of a eukaryotic cell; the slime mould Dictyostelium discoideum embedded in vitrified ice. Xy slice of a tomographic reconstruction from a complete tilt-series (120 images) of an approximately 200 nm thin peripheral region of the cell. Visualization by segmentation. Large macromolecular complexes, e.g. Ribosomes are shown in a green color, the actin filament network in orange-red and the cells’ membrane in blue. Cryo-tomograms of Dictyostelium discoideum cells grown directly on carbon support films have provided unprecedented insights into the organization of actin filaments in an unperturbed cellular environment. (Medalia et al. Science 298, 2002).
Lower row: Cryo-ET in combination with the single particle approach of transport-competent Dictyostelium discoideum nuclei. Three-dimensional reconstruction of the peripheral rim of an intact nucleus. X-y slice of 10 nm thickness along the z axis through a typical tomogram. Side views of nuclear pore complexes (NPCs) are indicated by arrows. Ribosomes connected to the outer nuclear membrane are visible (arrowheads). Surface rendered representation of a segment of nuclear envelope (NPCs in blue, membranes in yellow). Structure of the Dictyostelium NPC after classification and averaging of subtomograms (Beck et al.; Science, 306, 2004; Nature 449, 2007).
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