ABSTRACT
The coexistence of multiple distinct structural states often obstructs the application of three-dimensional cryo-electron microscopy to large macromolecular complexes. Maximum likelihood approaches are emerging as robust tools for solving the image classification problems that are posed by such samples. Here, we propose a statistical data model that allows for a description of the experimental image formation within the formulation of 2D and 3D maximum-likelihood refinement. The proposed approach comprises a formulation of the probability calculations in Fourier space, including a spatial frequency-dependent noise model and a description of defocus-dependent imaging effects. The Expectation-Maximization-like algorithms presented are generally applicable to the alignment and classification of structurally heterogeneous projection data. Their effectiveness is demonstrated with various examples, including 2D classification of top views of the archaeal helicase MCM and 3D classification of 70S E. coli ribosome and Simian Virus 40 large T-antigen projections.
Subject(s)
Antigens, Polyomavirus Transforming/chemistry , Archaeal Proteins/chemistry , Cryoelectron Microscopy/methods , DNA Helicases/chemistry , Imaging, Three-Dimensional , Models, Molecular , Ribosomes/chemistry , Algorithms , Antigens, Polyomavirus Transforming/ultrastructure , Archaeal Proteins/ultrastructure , Cryoelectron Microscopy/statistics & numerical data , DNA Helicases/ultrastructure , Escherichia coli/metabolism , Likelihood Functions , Models, Statistical , Protein Conformation , Ribosomes/ultrastructureABSTRACT
Although three-dimensional electron microscopy (3D-EM) permits structural characterization of macromolecular assemblies in distinct functional states, the inability to classify projections from structurally heterogeneous samples has severely limited its application. We present a maximum likelihood-based classification method that does not depend on prior knowledge about the structural variability, and demonstrate its effectiveness for two macromolecular assemblies with different types of conformational variability: the Escherichia coli ribosome and Simian virus 40 (SV40) large T-antigen.
