Electron tomography (ET) is a method for three-dimensional (3D) reconstruction of objects from a series of projection images (tilt series) recorded with a transmission electron microscope. The method is useful for imaging large macromolecules and supramolecular objects that cannot be obtained in regular arrays. Furthermore, the individual objects can be studied in si tu in relation to other components of the cell. Presently, ET covers the intermediate resolution range of 2.5-5.0 nm. The ET method complements established methods appropriate to reveal the atomic structures of isolated molecular constituents, such as X-ray crystallography. It is expected that ET will prove useful in the molecular analysis of complex structures such as spliceosomes and nuclear pore complexes. In Martinsried, procedures were developed to record tilt series automatically of vitrified specimens at an electron dose where structural details are preserved at atomic-resolution. In Stockholm, a method was developed (constrained maximum entropy tomography, COMET), that permits refinement of 3D reconstructions with low noise level. -- To develop the ET methodology and instrumentation towards a resolution better than 2.5 nm and to be able to handle large supramolecular objects, the following issues will be addressed. 1) The industrial partners Philips and TVIPS will provide advanced instrumentation optimized for ET which is not commercially available: electron microscopes equipped with field emission gun, in-column energy filter and CCD camera will be installed in Martinsried and in Stockholm.
2) The Martinsried group will develop the automated data collection procedures to increase the speed of data collection by a factor of five, and to improve the user-friendliness and applicability. 3) The Stockholm group will improve the performance of the COMET procedure to accommodate large (5123) 3D reconstructions. 4) The industrial partner Silicon Graphics Europe will develop computer graphics tools for interactive quantitative analysis, presentation and exchange of 3D reconstructions as well as software tools closely coupled to special hardware to increase the speed of the 3D algorithms by more than two orders of magnitude. 5) ET will be applied to the supramolecular structures studied in Martinsried (proteasomes) and Stockholm (a pre-messenger RNP particle), and the various technical improvements will be evaluated. The quality of the 3D reconstructions will be compared with the highresolution structural information available by e.g. X-ray crystallography for parts of the assemblies. Preparative procedures to apply ET to supramolecular assemblies in si tu will be further evaluated and developed in parallel with the analysis of the structures.
Funding SchemeCSC - Cost-sharing contracts
171 77 Stockholm
5600 MD Eindhoven