Three-dimensional Reconstruction from Electron Micrographs of Randomly Oriented Macromolecules. Knowledge of the structure of macromolecules is essential to an understanding of their functions in biological systems. Many large macromolecules can only be analyzed by threedimensional electron microscopy. For single asymmetrical particles, in contrast to crystalline objects, resolutions achieved in three dimensions have been limited to 2-3 nm. The techniques proposed here will contribute to a fuller usage of information contained in a micrograph and are an important step toward achieving higher resolution and thus a better understanding of biological function. A method is proposed for three-dimensional reconstruction from electron micrographs of randomly oriented single macromolecules. Biological particles are radiation sensitive, and imaging must be done under low-dose conditions. Only techniques that combine information of many identical particles can overcome the limits imposed by radiation sensitivity. One of the most successful methods of this kind was SECReT (Single Exposure Conical Reconstruction Technique), developed by the P.I.. Programs for this method have been distributed to laboratories world wide and have been used for the determination of more than 15 structures over the last seven years. However, SECReT requires that all particles used in a reconstruction have the same orientation within the plane of the specimen. Deviations from this preferred orientation limit the resolution in three dimensions. Proposed here is a new method for determining the orientation of particles from their projections. This technique can be used for refining the orientation of projections used for SECReT and for the determination of projection angles of particles with unknown orientations. Successful development of this technique will lead to substantial reduction in experimental effort and increase the resolution achievable in three dimensions. The theory of Radon t ransforms is the basis for three-dimensional reconstruction algorithms. Most research has focused on the design of efficient inversion algorithms and only a few applications have made use of their potential as image processing tools. In this proposal Radon transforms are used as tools for simultaneous translational and rotational alignment of projections in three dimensions. Once the orientations of the projections are known a threedimensional reconstruction can be calculated. The alignment/reconstruction method will be combined with a Gibbs sampling technique for overcoming noise limitations and for image classification. Programs to assess the feasibility of the proposed technique have been written and first results have been published. The new technique will be applied to two objects, the calcium release channel from sarcoplasmic reticulum and the 50S ribosomal subunit from Escherichia coli. The two macromolecules were chosen because they exhibit features that are advantageous for testing the procedures, and because results at higher resolution will contribute to a better understanding of their function. The four-fold symmetry of the calcium release channel can be used to increase the signal to noise ratio, whereas the 50S ribosomal subunit is a globular structure with few protuberances and ideal for testing the limits of the applicability of this method.
