Cryo-EM

The cryo-EM facility at the Department of Structural Biology in the basement of BST3 is furnished with state-of-the-art electron microscopy instrumentations and has full capability to carry out high resolution three-dimensional structural analysis of proteins, protein complexes, phages and viruses, macromolecular assemblies, cellular organelles, and bacterial cells. It comprises three electron microscopes from FEI: a Polara G2 electron microscope operating at 300kv with a cartridge based helium specimen stage and a field emission gun (FEG), equipped with a high-resolution 4kx4k CCD camera; a Tecnai F20 electron microscope operating at 200kv with a liquid nitrogen specimen stage, also with a FEG and a high resolution 4kx4k CCD camera; and a Tecnai T12spirit operates at 120kv with a LaB6 thermo filament as the electron source. The first two scopes are capable for low-dose cryo-EM and electron tomographic studies, especially the Polara G2 with a super stable cartridge based stage design. The T12spirit is for room temperature specimens and has features of automatic tuning and alignment. The facility is also equipped with a FEI vitrobot for frozen-hydrated specimen preparation and other ancillaries for cryoEM. The cryo-EM facility is managed by Dr. Alexander Makhov.

The two faculty engaged in cryo-EM methods are James Conway (single particle studies) and Peijun Zhan (electron tomography).

Conventional electron microscopy provides two-dimensional (2D) projection images of the specimen, which are summation of the specimen information along the electron beam path. By combining information from series of 2D projections representing different views of the specimen, it is also possible to extend electron microscopic imaging into the third dimension. Emerging methods in 3D biological electron microscopy provide powerful tools to explore the internal 3D architectures of tissues, cells, organelles and macromolecular complexes. It bridges a critical gap in imaging in the biomedical size spectrum, the gap between molecules at atomic resolution by X-ray and NMR and cells and tissues by light microscopic and conventional electron microscopic methods. This gap comprises a size range of considerable interest in biology and medicine that includes cellular protein machines, large protein assemblies and nucleic acid assemblies, small subcellular organelles and small bacteria. These objects are generally too large and/or too heterogeneous to be investigated by high resolution X-ray and NMR methods; yet the level of detail afforded by conventional light and electron microscopy is often not adequate to describe their structures at resolutions high enough to be useful in understanding the chemical basis of biological function.

Two areas in 3D electron microscopy are of particular note: (I) "single particle" microscopy and (II) "electron tomography".

1. "single particle" microscopy
   Biological specimens are radiation sensitive, resulting in poor signal-to-noise ratios in electron microscopic imaging. To overcome this problem, images recorded from thousands of identical copies of specimens randomly oriented relative to the electron beam are combined and averaged. This method is referred as "single particle" microscopy. A variety of 3D structures have been determined at resolutions in the range of 3–30 Å , which allows interpretation of complex structures in terms of their underlying tertiary, secondary and primary structural elements. This methodology can be used to determine the structures of complexes when they are too large and/or too heterogeneous to be investigated by high resolution X-ray and NMR methods. The structure will provide the molecular envelope for the large complexes into which the atomic structure of individual components determined either by X-ray crystallography or NMR can be docked, and the interpretation on the interactions within the HIV/host complex cab be achieved at near-atomic level.
   
   
2. Electron tomography
   Electron tomography (ET) involves structure determination of "one-of-a-kind" objects such as whole cells and subcellular organelles, in which averaging methods are generally not applicable. The underlining principle is similar to computerized tomography (CT). Instead of rotating the radiation source and detector around the object, in ET, the specimens are tilted around the incident electron beam and a series of projection images over a range of orientations are recorded and combined computationally to reconstruct the 3D structure of the object. A fundamental limitation to the studies of whole-cell specimens is that useful images can only be recorded from the thinnest regions of the cell (typically <1 µm). This method can be applied to investigating whole cells. Three approaches may be employed in cellular studies. (i) ET of fixed and stained specimens at room temperature. The cells can be fixed (cryo or chemically) and embedded and sectioned. This has been a successful approach used to yield information on the membrane connectivity of cells and organelles or for recognition of cellular location of large and easily-stained objects. (ii) Cryo-ET of unstained specimens rapidly vitrified at liquid nitrogen temperatures. This approach allows obtaining structural information about protein complexes and cellular assemblies at near native state of the cell.