Unlike traditional electron microscopy, which uses thin sections to visualize the structure of cells or tissues, molecular electron microscopy is used to determine the three-dimensional structure of individual proteins or protein complexes. To achieve the much higher resolution required for the visualization of a protein structure, molecular electron microscopy takes advantage of low-dose imaging techniques to reduce beam damage of the specimen and computational averaging techniques to improve the signal-to-noise ratio. The two main techniques used in molecular electron microscopy are electron crystallography and single particle averaging. In electron crystallography, the protein is crystallized in two dimensions and the structure determined using diffraction techniques. This approach is particularly powerful for membrane proteins and can yield an atomic structure for the protein. In the single particle averaging approach, individual molecules are imaged and the structure determined by back projection algorithms in real space. Although the resolution of the resulting density maps is typically only around 25 Å, single particle methods can yield structural information for macromolecular complexes that are often very difficult to solve by X-ray crystallography. Particularly powerful is the combination of molecular electron microscopy with X-ray crystallography for macromolecular complexes, where the atomic structures of individual components, as determined by X-ray crystallography, are fit into the molecular envelope of the entire complex determined by electron microscopy.
Equipment
The molecular electron microscopy facility centers around two state-of-the-art Philips Tecnai electron microscopes, a 120 kV instrument equipped with a Gatan 1k x 1k charge-coupled device (CCD) camera and a 200 kV instrument with a field emission electron source and a Gatan 2k x 2k CCD camera. Both electron microscopes are equipped with a Gatan low-light camera, compustage, cryo-holders, anti-contamination blades, spot-scan and low-dose software. The electron microscopy suite also includes a dark room to develop electron micrographs and a small laboratory with specific equipment for specimen preparation such as carbon evaporator, glow-discharger, and plunger for the vitrification of biological specimens. In the near future, we will also have access to a Tecnai F30 electron microscope operated at 300 kV and equipped with a helium specimen stage. This instrument will be located at Brandeis University as part of a shared facility between Harvard and Brandeis University.
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Philips Tecnai 12 electron microscope This instrument is our workhorse, which is mainly used for negative stain and low-resolution cryo-work. |
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Philips Tecnai F20 electron microscope With its 200 kV acceleration voltage and its field emission gun, this instrument is well suited for high-resolution imaging. |
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A dedicated computer room houses all the equipment necessary for digital processing of electron microscopic data, especially a JEOL JFO-3000 laser diffractometer and a Zeiss SCAI scanner.
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JEOL JFO-3000 laser diffractometer The laser diffractometer is used to evaluate the quality of our electron micrographs and, in the case of two-dimensional crystals, to find well diffracting areas. |
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Zeiss
SCAI scanner The scanner is used to digitize our electron micrographs to make them available for digital image processing. |
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Computer Room The computer room houses several Dec Alpha and SGI workstations, on which image processing software is run. |
Our Commitment
The molecular electron microscopy facility at Harvard Medical School is predominantly used for our own research (Walz lab web site) and can therefore not be considered a service facility. We are, however, committed to make our technology available to as many research groups in the community as possible. If you are interested to use molecular electron microscopy, please contact us by phone or email.
Contact
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Thomas WalzGroup Leader Department of Cell Biology Phone: 617- 432-4090 |
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Zongli LiManager Department of Cell Biology Phone: 617-432-4095 |