Three‐dimensional reconstruction from a single‐exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli

Abstract

We present a new reconstruction method that takes advantage of the fact that many biological macromolecular assemblies show a preferred orientation with respect to the plane of the specimen grid in the electron microscopic preparation. From one micrograph taken of such a specimen tilted by a large angle, a conical tilt series with random azimuthal angles can be extracted and used for a three-dimensional reconstruction. Our technique allows the determination of the molecular structure under low-dose conditions, which are not achievable with reconstruction methods that use conventional tilt series. The reconstruction method combines a number of existing image processing techniques with a newly developed weighted back-projection algorithm designed for three-dimensional reconstruction from projections taken with arbitrary projecting directions. The method is described as it was applied to the three-dimensional reconstruction of the structure of the 50S ribosomal subunit of Escherichia coli (E. coli).