Research
Electron Tomography (WP4, WP5, WP6, WP8, WP13)
Electron Tomography (ET) is the method of choice to obtain three-dimensional images of large cellular structures, such as organelles or even whole cells, at molecular resolution (2-4 nm). This method resembles the Magnetic-Resonance-Tomography (MRT) - regularly used in the clinical praxis - in that multidirectional images are taken from one object. The basic principles of ET have been known for years but its application to sensitive biological material has been difficult due to the highly damaging radiation doses of this method. Recent technology advancements rendered an automated data acquisition possible and thus allowed the reduction of the total electron dose.
Embedding of the biological specimen in vitreous ice enables studies of the macromolecular organization in the cell. Whole prokaryotic and small eukaryotic cells can be directly grown and hydrated frozen on Electron Microscopy-grids. Examination of the - thus naturally preserved - cells via cryo-ET delivers images of the cellular structures in their functional environment. Such so called "tomograms" contain all information about spatial relationships of macromolecular structures in the cell. Due to their poor signal-to-noise ratio and the generally highly crowded nature of the cytoplasm the interpretation of these tomograms remains difficult. To get significant information about specific structures in the cell, the images have to be evaluated using advanced pattern recognition methods. Already existing structural models of cellular structures with lower resolution can help to examine the tomograms systematically. The final aim will be to visualise the complete three-dimensional composition of the cell at molecular resolution.
Single Particle Analysis (WP9, WP10, WP11)
Developed in the last two decades the Single Particle Analysis (SPA) provides - additionally to for example Electron Crystallography or Cellular Tomography - a powerful tool to study biological macromolecules. Using the traditional tomography, a series of tilted images of one object is recorded and finally integrated into one three dimensional model. In contrast SPA requires a large number of identical copies of the object of interest that are frozen in their natural state and in random orientations on EM (Electron Microscopy) grids. With one electron dose delivered in a single projection thousands of these identical molecules are imaged individually. Each copy provides a unique projection direction. Determination of their relative orientation and aligning all these images computationally allows a three dimensional reconstruction of the object of interest.
One of the limiting factors in the SPA is the number of individual molecules. To obtain high-resolution reconstructions, more than 100,000 well-purified particles are necessary. Another limitation is the size of the molecule: theoretically an object as small as 100 kDa can be analyzed - if the conditions are idealized (Henderson, R. Quart. (1995). Rev. Biophys. 28, 171-193). But molecules in the range of 1-10MDa seem to be much better suited. The method has been successfully applied to large symmetric complexes such as the chaperonin system as well as to asymmetric macromolecular assemblies like ribosomes. The high advantage of the SPA is - in contrast to for example the Electron Crystallography - that a regular arrangement of the individual particles is not necessary.
Electron Crystallography (WP7)
Using electrons instead of X-rays or neutron diffraction Electron crystallography (EC) opens up new possibilities for the crystal structure determination of biological macromolecules. EC works with well-ordered two dimensional crystals of proteins that require less material and form more readily than single three dimensional crystals. Thus the technique of EC appears particularly suited for complex membrane proteins since it helps to overcome two of the main difficulties in the crystallization of these proteins. In addition, EC can be applied to smaller samples than X-ray diffraction due to the strong interaction of electrons with matter.
The regular arrangement of the biological molecules in the crystal enables the direct averaging of the information - obtained from several hundreds or thousands of particles - in a fourier transform. The reconstruction of the object of interest is achieved by inverse Fourier or Fourier-Bessel transformation.
Starting with the work on bacteriorhodopsin in 1975 which led to the first visualization of the secondary structure of a membrane protein (R. Henderson and P. N. T. Unwin (1975). Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257, 28-32) EC has made significant progress in the last decade due to the development of new equipments and software - tailor-made for this crystallization technique.
Instrumentation and Software (WP2, WP3)
Structural analysis by single particle analysis, electron crystallography and electron tomography still is slow compared to other structure-determination technologies, in particular x-ray crystallography. Processing time typically is in the range of several months per solved structure, depending on the resolution achieved, whereas it is in the range of hours or days for x-ray crystallography, once suitable crystals are available.
A joint effort of the research community and manufacturers to develop user-friendly, universal interfaces between electron crystallography, single particle analysis and electron tomography will reduce the waste of resources in redundant activities. In a first stage, 3D-EM will work towards a unified data format and a supported user interface to help users move easily between the differ-ent software systems available. This will also include testing of software and consistency between different packages.
In a second stage, an optimised standard software platform will be established, which will reduce processing times in image reconstruction and facilitate the integration of data obtained with dif-ferent methods. The "Image Processing Tools" (ipt) initiative may serve as platform to reach that goal.
In a third stage, the modelling and refinement procedures will be standardized. Although the level of resolution addressed imposes different requirements, the common principle is to exploit all the structural information available to establish a model and to test it against the data collected with the electron microscope. Software to facilitate and accelerate this step will thus exhibit similar features.
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