NYSBC - New York Structural Biology Center

Overview of EM Techniques

General Information

The 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 microscopy

The 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.

A. Single particle analysis

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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.

B.Electron crystallography


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).
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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.
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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

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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).
For samples that are neither homogeneous nor symmetric, the 3D structure can be obtained using procedures related to medical CAT scans. Similar to X-rays, each electron micrograph provides a projection image of the object being studied. By collecting images with small angular increments, it is possible to determine the 3D arrangement of material within the sample volume. Electron tomography has proven valuable for visualizing mitochondria and cell junctions (figure 4) within tissue sections or asymmetric surface projections in frozen suspensions of bacteriophages. We have also had recent success imaging cultured cells that have been frozen directly on the EM support. Data collection is automated using the SerialEM software package (Mastronarde (2005) J. Struct. Biol. 152:36). Although each microscope has a unique user interface, the use of SerialEM provides a uniform interface for tomography, facilitating migration from one machine to another. For 3D reconstruction we use IMOD for samples which contain fiducial markers (Kremer (1996) J. Struct. Biol. 116:71) and protomo (Winkler H. (2007) J. Struct. Biol. 157:126) for those that do not (e.g. frozen-hydrated samples).

Facility and resources

Instrumentation

To enable the application of the above-mentioned techniques to a variety of biologically relevant samples, the NYSBC has five electron microscopes for use by affiliate members: A 120kV screening microscope (JEOL 1230); two 200kV cryomicroscopes (Tecnai F20 and JEOL 2100); a 300kV energy-filtered cryomicroscope (JEOL 3200); and a dual beam FIB/SEM (Helios Nanolab 650). The FIB/SEM, 200kV and 300kV instruments have field emission guns. All microscopes have computer interfaces that communicate with specialized programs for automated acquisition of data, which is convenient for single particle analysis and crystallography and absolutely essential for tomography. In addition to the microscopes, we have all necessary equipment for sample preparation, such as two plungers to freeze crystals or macromolecular suspensions, two high pressure freezers for tissue, a freeze substitution machine, two ultramicrotomes, a carbon evaporator for making sample support films and a wide variety of negative stains. Thus, affiliates simply need to provide suitable samples and all EM-specific sample preparation can be handled at NYSBC. For further details see equipment.

Technical assistance

In addition to instrumentation, affiliates can take advantage of NYSBC staff expertise in each of these four technologies. Initially, staff will discuss the feasibility of projects and lay out a plan for the different stages of a proposed research project. Affiliates can work closely with NYSBC staff to evaluate the suitability of existing samples for achieving specified goals and in implementing any unusual experimental approaches. Staff members are also available for pilot studies to produce preliminary reconstructions suitable for inclusion in grant proposals. A sustained commitment from the affiliate lab is generally required to achieve the ultimate goal and NYSBC staff routinely provides training to individual researchers to be able to function in the supported environment at NYSBC. At the beginning of a project, this involves plunging samples into liquid ethane, transferring these samples into the microscope column and collecting the necessary images. Microscopes are aligned each morning and staff is available for assisting in sample loading and microscope operation. Once affiliates obtain a basic understanding of operation, a training course is used to establish independence, thus making microscopes available 24/7. NYSBC offers a course each fall semester in image processing in the fall semester in which leading researchers from the NY area together with NYSBC staff teach the principles and practice of 3D structure determination by electron microscopy. All major software packages are installed at NYSBC and continuing guidance in their use is available through personal interactions with our staff. For further details see staff.