3D Reconstruction of Proteasomes Using Random Conical Tilting

Abstract

Proteasomes isolated from the archaebacterium Thermoplasma acidophilum and negatively stained with ammonium molybdate appear in two preferred orientations on electron micrographs. Describing the particle roughly as cylinder-shaped, the orientations "end-on" and "side-on" are defined by the cylinder axis being normal and parallel to the specimen support, respectively. Two-dimensional averages are available from both views. The complexity of the particle, however, prevents an intuitive deduction of a three-dimensional model from these two views. Due to the occurence of two preferred orientations, the object is, in principle, well suited for a 3D reconstruction based on random conical tilting. The side-on view was used for 3D reconstruction for two reasons: First, two-dimensional averages led us to suppose that the particles do not suffer from rotations around their cylinder axis, and second, parallelism of the long cylinder axis and the specimen support promises to give a stable object inclination.The proteasomes were isolated and prepared for electron microscopy as described previously. Many pairs of micrographs were taken in a Philips EM 420 at a magnification of 36000 and an electron dose of about 2000 e/nm2. The first exposure of each pair shows the specimen with a tilt angle of about 60 deg, the second one is untilted (Fig. 1). Several image pairs were selected for computer processing using a favourable distribution of particles and acceptable focus conditions as selection criteria. After digitizing corresponding areas (2048 by 2048 pixels, pixel size 15 μm = 0.42 nm at object level) in each pair using an Eikonix 1412 camera, small images of single particles were extracted from the untilted image and aligned with respect to translation and rotation. Knowing the tilt angle and tilt axis azimuth, position and rotation parameters of the particles could be transferred to the tilted image thus enabling the extraction of a set 227 of projections ready for a three-dimensional back projection (Fig. 2). All computation was done with the EM-system.