Most structural techniques examine the average properties of a very large number of molecules. For example, a .1 mm cubic crystal of a 10nm particle would contain approximately 1 trillion individual protein molecules aligned in a crystalline lattice. When x-rays diffract from this crystal, the positions and intensities of the resulting spots can be used to determine the structure of an individual protein molecule, often to atomic resolution (although the difficult 'phase problem' still exists). In single particle analysis, the molecules to be studied are prepared in a solution which is then frozen in a very thin (~1um) layer of vitreous ice. This ice is frozen so rapidly it does not crystallize, it remains in a fluid-like conformation at liquid nitrogen temperatures. This preserves the protein in its native fluid conformation. This sample is then placed in a transmission electron microscope where images of the individual molecules in random orientations are collected. These images represent projections through the molecule. The individual molecule images are then processed in a computer to determine the 3D structure of the molecule. A typical reconstruction uses 10,000 - 100,000 particle images. Note that this is an extremely small amount of protein, much less than is required to prepare a crystal. Currently (1999) this technique is limited to ~7 angstrom resolution, but microscopes and analysis techniques are rapidly advancing. It is likely that near atomic resolution will eventually be achieved.
This techniques has several advantages over x-ray crystallography: 1) No phase problem. Electron microscopy implicitly measures phases. 2) In x-ray crystallography, smaller samples are easier to crystallize and process, in cryo-EM, larger molecules are generally easier. Particles as large as 2000 angstroms have been processed using this technique. X-ray crystallography tends to have problems for particles larger than 100 angstroms or so. 3) Very often a protein can exist in one of several different functional states. Usually it is very difficult to crystallize a protein in each of these states to determine the structural changes involved. However, using single particle analysis, it is often possible to statistically analyse the particles and separate the functional states from the overall population. If this proves difficult, the protein (in solution) can be biochemically driven into a nearly homogenious population of a single functional state. This solution is then frozen and imaged.
The Software
EMAN consists of a set of image processing tools which are specifically
designed to make the single particle reconstruction technique easy to perform.
It also contains a variety of useful routines for general image processing,
as well as a complete C++ library of routines useful in analyzing electron
micrographs. EMAN was designed with a 3-tier structure. The top tier consists
of programs with GUIs (graphical user interfaces) which allow data browsing,
analyzing the results, and hand-holding the user through the reconstruction
process. In the middle tier are command-line programs which perform the
actual reconstruction, eg - a program to generate projections of a 3d model,
another to classify particles based on a set of reference images, etc.
In the lowest tier is a set of C++ objects and C functions which allow
transparent EM file access, fourier transforms, filtering, etc.
EMAN was designed to automate as much of the single particle reconstruction process as possible. In general, once a set of boxed out particle images have been prepared, the entire reconstruction proceeds with almost no human intervention. When the run is done, a complete set of tools for analyzing the results are provided. An initial, low resolution reconstruction of a new protein can typically be processed overnight on a single processor P-II class computer. For example, ~2000 particles of Ca++ release channel at 80x80 pixels/particle were processed to produce a ~28 angstrom 3d model in about 8 hours on a single processor SGI Octane (comperable to a PII - 400). This is compared to approximately 3 weeks of user-intensive work with Imagic to produce the same result.
IMPORTANT NOTE: EMAN does incorporate features for performing reconstructions of particles with icosahedral symmetry, but it was really designed with less symmetric particles in mind. The icosahedral routines have not been tested and optimised very well yet. They are likely not to work very well at this point. You're welcome to try it, but be aware that it's not really ready for this use yet.
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