Three-dimensional (3D) structure of a wide range of biological macromolecular assemblies can be computed from two-dimensional (2D) images collected by transmission electron microscopy (EM). This information integrated with other structural data (e.g., from X-ray crystallography) helps structural biologists understand the function of macromolecular complexes. Single-particle analysis (SPA) is a method used for studies of macromolecular assemblies whose structure and dynamics can be analyzed in isolation (e.g., proteins, ribosomes, viruses). It is complementary to nuclear magnetic resonance since it allows computing the structure of large assemblies (diameter of 10-30 nm). It is also complementary to X-ray crystallography since it allows studying non-crystalline matter.
Typically SPA uses a large number of images of randomly oriented individual molecules to reconstruct the 3D structure. In practice, the analysis requires EM images of thousands of individual molecules of the same protein captured in a unique conformation taken in random orientation. When the distribution of single-particle orientations samples Fourier space completely and the population is homogeneous, standard image processing strategies allow computing an average structure at resolution of 0.4-1 nm.
In addition, SPA has shown to be promising in capturing alternative conformations of the same macromolecular complex. To understand the functions of macromolecular assemblies, it is often necessary to elucidate their structural dynamics by determining these alternative conformations. SPA is well suited for studying dynamics of large macromolecular complexes that display multiple conformations in solution since images of individual molecules can be potentially sorted computationally.
However, in the framework in which the conformational change is considered as a continuous movement, any kind of classification (in 2D or in 3D) samples the continuous movement into discrete, average conformations which limits the resolution of the computed conformations meaning that some different but close conformations may not be detected. The techniques for the analysis of molecular dynamics from EM images are currently under permanent development and may provide strong impact on structural biology and drug discovery. This talk is about a recently developed method that combines normal mode analysis with volume-to-image registration for studying continuous-type conformational variability.