Overview of EM Techniques
General InformationThe goal of the NYSBC electron microscopy facility is to help researchers elucidate the intermolecular interactions and domain architectures of macromolecules within their native cellular assemblies. Towards this goal, the facility has brought together a combination of instrumentation and staff expertise that supports the determination three-dimensional structures using the major techniques available to the field.
Brief overview of electron microscopyThe first three techniques ( a) single particle analysis, b) electron crystallography and c) helical reconstruction) are applicable to samples that can be biochemically isolated; these samples require high purity as well as a high degree of structural homogeneity. These are the techniques that produce the highest resolution, which can approach atomic resolution in favorable cases. Fitting atomic models to the resulting structures is a common approach for interpreting the resulting structures and deducing interactions between domains or subunits of a larger assembly. Electron tomography, in contrast, can be used to visualize highly-complex and heterogeneous samples, such as tissue sections, or pleomorphic assemblies such as liposomes. Since there is often no aid from innate symmetry, the resolution achieved with electron tomography is lower, but still sufficient for evaluating the topology of organelles or distributions of macromolecular assemblies across the surface of a virus.
|Figure 1. Three-dimensional reconstruction of pol-gamma using single particle analysis. Two subunits with known structure have been fitted to the electron density map determined by cryo-EM.|
Large macromolecular assemblies (>300kDa) are generally challenging to crystallize. Electron microscopy is well suited for structure determination of such complexes, assuming a homogeneous preparation in which all particles in the preparation have identical shape and composition. The ribosome and GroEL are classic examples, the former providing reconstructions at better than 1 nm resolution. For three-dimensional reconstruction, single particle analysis typically requires aligning and averaging several thousand images, and considerable effort is usually required for data collection. In addition, computationally intensive refinement is required to obtain the best result. NYSBC staff members have experience with several software packages, and are actively involved in helping researchers evaluate their samples and process the resulting images.
A two-dimensional crystal array is commonly formed by membrane proteins. Typical examples are bacteriophodopsin and the calcium ATPase (figure 2). Due to their small thickness, these crystals are not amenable to structural studies by X-ray crystallography, but suitable methods to extract structural information using cryo-electron microscopy have been developed since the 1970's. The instruments at NYSBC have all the technological requirements to collect the best possible data as well as software for processing 2D crystal data (e.g. 2dx software package: Gipson et al. (2006) J. Struct. Biol. 157:64).
|Figure 2. Image of a two-dimensional crystal of Ca-ATPase in the background, with the atomic model of the molecule superimposed.|
C. Helical reconstruction
Helical symmetry is ubiquitous in nature, as it allows the formation of large assemblies using regular contacts between a single type of protein molecule. Helical symmetry can be found in filamentous viruses (e.g., Pf1), in proteins of the cytoskeleton (actin, tubulin), or in proteins that form two-dimensional crystals folded onto the surface of a cylinder, such as the acetyocholine receptor or CopA (figure 3). One of the advantages of helical symmetry is that a single assembly has enough different views of the constituent molecules to provide a three-dimensional reconstruction. Software for extracting three-dimensional information from images of helical particles has been implemented at NYSBC and our staff is experienced both in imaging and in analyzing these samples.
|Figure 3. Tubular crystal of an ion pump is shown on top and a 3D reconstruction determined using the helical symmetry during image processing is shown below.|
D. Electron tomography
|Figure 4. Section through an electron tomogram of a cell junction, showing segmentation of the cell membrane (cyan) and protein connections to the intermediate filament network (blue).|