1) Two-dimensional crystals. This type of crystalline array is commonly formed by membrane proteins. Typical examples are bacteriophodopsin and the calcium ATPase (figure 1). 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). back to top

Figure 1. Image of a two-dimensional crystal of Ca-ATPase in the background, with the atomic model of the molecule superimposed.

2) Particles with helical symmetry. 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 2). 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. back to top

Figure 2. Tubular crystal of an ion pump is shown on the left and a 3D reconstruction determined using the helical symmetry during image processing is shown on the right.

3) *Single particle analysis*. Large protein assemblies (>300kDa) are generally challenging to crystallize. Nevertheless, electron microscopy can be used for structure determination, 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 supports two packages for 3D reconstruction: SPIDER (Frank J. et al. (1996) J. Struct. Biol. 116:190) and EMAN (Ludtke et al. (1999) J Struct Biol 128, 82) that have been implemented on our computational cluster in order to speed up these refinements. NYSBC staff members have experience with both of these software packages, and are actively involved in helping researchers evaluate their samples and process the resulting images. back to top

Figure 3. 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.

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).

4) Electron tomography. 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). back to top