THREE DIMENSIONAL RECONSTRUCTION WITH CONTRAST TRANSFER COMPENSATION FROM DEFOCUS SERIES

Abstract

Cryo-electron microscopy provides the means to quantitatively study macromolecules in their native state. However, the original mass distribution of the macromolecule is distorted by the contrast transfer function (CTF) of the electron microscope. In addition, the zeros of the CTF put a practical limit on the resolution that can be achieved. Substantial improvement to the quality of the results can be accomplished by collecting the data using a series of defocus settings. Such data sets can be combined and the resolution can be extended beyond the first zero of the CTF. This procedure can be applied either at the stage of raw data, or more effectively at the stage of reconstructed volumes which have a high signal-to-noise ratio as a result of averaging over many projections. A method of threedimensional (3D) reconstruction that combines an algebraic, iterative 3D reconstruction technique with CTF correction is proposed. The potential to incorporate a priori knowledge into the reconstruction process is discussed. This approach was used to obtain a 3D reconstruction of the E. coli 70S ribosome from energy filtered cryo-images.