Electron microscopy

Transmission Electron Microscopy – Structural analysis of Protein complexes

 

Transmission Electron Microscopy (TEM) is an established technique to analyze the structure of thin samples. TEM projects the image of protein complex particles, viruses or cells deposited on a thin film or embedded in a thin layer of frozen ice (Cryo-TEM) onto a detector (like a photographic film or a camera). The projections of the objects will be reconstructed in silico to obtain a tridimensional structure. Hence, crystallization is not a prerequisite to determine the structure of macromolecular complexes. Optimal results using TEM can currently be performed on complexes that are 250kDa or larger. However, this limit is being pushed downward by new technology such as direct electron detectors. As TEM enables visualization of a wide range of particles in solution, it complements the biophysical toolbox available from NovAliX.

 

Negative Staining Transmission Electron Microscopy

Single particle analysis (SPA) using Negative Staining Transmission Electron Microscopy is a powerful technique to characterize the morphological features of protein complexes under specific circumstances (like epitope mapping) at intermediate resolution (20Å and above). The sample is deposited on a thin layer of carbon and surrounded by stain. As this staining is usually made of heavy metal salt, it will provide a very high contrast and is less radiation sensitive than biological matter. The sample preparation and data acquisition can be performed at room temperature and the required concentration is one to two orders of magnitude lower than X-Ray crystallography and NMR, although the target molecules need to have a minimum size. There is no limitation in terms of maximum size. Similar particles can be classified in groups and averaged together to improve the signal to noise ratio. If the protein complexes don’t show a preferred orientation, 3D reconstruction can be performed at intermediate resolution to generate an initial model. Such model is usually the starting structure for a more advanced 3D Cryo-TEM Single Particle Analysis (see below).

 

3D Cryo-Transmission Electron Microscopy Single Particles Analysis

Three Dimensional Cryo TEM Single Particles Analysis (3D cryo-TEM SPA) has been used to resolve tertiary structures of large proteins and quaternary structures of protein complexes down to the molecular and sometimes near-atomic resolution. Since crystallization or a high concentration of the sample are not mandatory prerequisites to obtain a good structure, Cryo-TEM is applicable to most proteins and provides direct observation of the mechanical dynamics and conformation of flexible structures close to their natural state. Because the particles are analyzed separately, the native structure is not distorted by non-natural circumstances. Hence, computational sorting of diverse conformations of proteins or protein complexes in a given preparation enable the visualization of various intermediate states and can provide kinetic information about a specific interaction between individual molecules. As XRD and NMR techniques report an average result based on the summed signal of a large population of individual molecules, such events might be overlooked. The main prerequisite for Cryo-EM is a homogeneous distribution of particles in a thin layer of ice. The potential initial model obtained from negative stain TEM (see above) can be a starting point for the structure refinement.

Recently, Cryo-TEM has moved to near atomic resolution through new developments in detectors (direct electron detection cameras), novel imaging techniques (de-blurring via movie mode of direct electron detectors), contrast enhancement (phase plates), minimal contamination environment, and electron optical aberration correction techniques. NovAlix provides access to such technology through its Cryo-TEM sample analysis program. Our area of expertise includes the stabilization of the biological complexes using advanced screening methods (ProteoPlex), cryo sample preparation suitable for high resolution TEM, acquisition of large data sets, 2D classification of particles and 3D reconstruction of models. If component structures solved by X-Ray or NMR are available, we will fit them into the 3D EM model density map to display structural features that could not be revealed by either technique separately.