News and Publications

hat is, until after they sat down with Professor Ron Milligan and drew up plans on a blank blueprint of the interior of the CarrAmerica B building.

In the following weeks, this became the blueprint for Milligan's dream of one of the most advanced biological microscopy centers in the world--The Center for Integrative Molecular Biosciences (CIMBio)--which officially opened in April. CIMBio is built around its advanced microscopes and open laboratories, and houses several TSRI faculty under one roof.

"We had an almost unique opportunity to design an ideal electron microscopy suite, and we put a lot of effort into doing this," says Milligan.

"It's quiet, there are no air currents, and the microscopes are sitting on a very stable platform," says Milligan.

MOLECULAR MACHINE MANIA

Milligan, Carragher, and Potter, all members of the Department of Cell Biology, are founding members of CIMBio, which was organized to combine the talents of several groups across campus who have backgrounds in divergent disciplines such as chemistry, biochemistry, structural biology and cell biology but whose interests converge in one area.

The center seeks to speedily obtain and analyze high-resolution structural images of large molecular complexes of the cell by combining the use of x-ray crystallography and electron microscopy (EM) as a means to unravel the structure and mechanism of action of the large molecular assemblies of the cell. These include the transcription complexes that make messages from the genes, membrane channels and pumps that import and export materials, and the tiny molecular tracks and motors that move cells and form important structures like the mitotic spindle.

Phase I of CIMBio is devoted to working out the structure of the proteins and nucleic acids in complexes that carry out the work of the cell. In addition to Milligan, Carragher, and Potter, CIMBio members involved in Phase I include investigators Francisco Asturias, M.G. Finn, Jack Johnson, Elizabeth Wilson-Kubalek, Tianwei Lin, Mari Manchester, Nigel Unwin, and Mark Yeager.

While the individual protein components of these cellular machines may be studied by x-ray crystallography, the machines themselves are compositionally and conformationally dynamic, making them often unsuitable for x-ray methods. They are, however, ideal specimens for electron microscopy. Polymerases, membrane complexes, viruses, and motor proteins can all be visualized in their native environment using EM. Also relocating to the new facility at the end of the year will be Geoffrey Chang, whose expertise in solving the high-resolution structures of integral membrane proteins is a valuable addition to the center.

Phase II will concentrate on the dynamics of cellular machines -- their assembly, disassembly, and control over time. Laboratory space for that effort is already under construction, and at the end of the year, investigators Velia Fowler, Klaus Hahn, Clare Waterman-Storer, and Kevin Sullivan will relocate there to lead the Phase II efforts.

The building combines several of these laboratories into large contiguous shared spaces built above and around the microscopes. The laboratories have an open design and some of the facilities--like the microscopes and an imaging area--are shared, something that the CIMBio researchers appreciate.

EM IMAGING OF BIOLOGICAL STRUCTURES

Electron microscopy, which has been around since the 1930s, uses a beam of electrons to image tiny objects onto a digital camera or a photographic plate. An electron microscope can examine specimens over a very wide range of magnifications, from no more than that which an ordinary microscope can produce, about 60 times, to incredible magnifications of up to 1,000,000 times.

The final products of these electron images are three-dimensional (3-D) maps of the cellular structures at near-atomic resolutions -- up to about 3 to 4 angstroms under the best of circumstances (an angstrom is one 254 millionth of an inch). When combined with the x-ray structures of the component parts of the structures, EM maps can yield a detailed description of the structure and action of the entire machine.

Further application of this technique will be an invaluable tool for studying membrane-bound proteins, which are notoriously hard to crystallize. Less than one half of one percent of the structures contained in the Brookhaven National Laboratory Protein Data Bank are of integral membrane proteins, despite the fact that over a third of all proteins in the body are in the membrane.

But EM can be a tedious technique. Calculating an EM structure manually takes weeks or even months.

A single high-resolution image of a sample under an electron microscope has too much noise to yield accurate molecular representation. Images must be averaged together with their counterparts to reduce noise. To build a 3-D map, one must take many images and build a structure by looking at all the different angles of all the different molecular assemblies imaged.

In this way, building a 3-D map is like looking at a piece of sculpture in a gallery. Only by walking around the piece and viewing its various sides and angles can the brain build a mental image of the art and fully comprehend its dimension, perspective, and scale. The same is true using a computer; only by piecing together many different views of a molecule from a microscope can a computer build a model of the molecular assembly.

And the molecule that is being imaged gets destroyed in the process, so the next image must be captured from some other part of the sample holder grid. This has always required a person to choose different spots on the grid manually. As the number of grid spots goes up, so goes the level of tedium.

"What we really want is 100,000 to 1,000,000 molecule images and that just takes too long to do manually," says Carragher. "Then you want to do 10 different conformational states, 20 different labeling studies, and each time it's going to take three to six months. That totals more than the lifetime of a graduate student."

Carragher and Potter, who lead the Automated Molecular Imaging Group, are creating algorithms for automated data collection and analysis, which should simplify the technique of electron microscopy and enable throughput to be increased dramatically.

AUTOMATION FOR EM

Several years ago, Carragher and Potter suggested that automated data collection and analysis could be developed for EM. A similar goal had been accomplished in x-ray crystallography; given the need for structural information in our post-genomics proteomics world, automation would represent significant progress.

They succeeded in developing software for both the collection and the analysis, which they brought to TSRI when they came last year to form the Automated Molecular Imaging Group at TSRI.

Creating the algorithms was not easy. Using the manual technique, a person has to make decisions about where to focus the EM beam and take a picture, looking first at low resolution and then deciding in which areas to collect data at high resolution. For automation to succeed, the computer must do the same thing and use intelligent criteria to search the low-resolution image for appropriate targets.

Carragher and Potter had to write their software to take a low-resolution image, select areas to image in medium resolution, and then analyze that image and strip out targets for high-resolution maps. Then, they had the computers put the data into processing programs and calculate 3-D maps. Recently, they have been testing and refining the programs.

"What we have done over the past year is to show that you can insert a sample in the microscope andcalculate a 3-D map fully automatically," says Potter.

In fact, Carragher and Potter constructed one of the best 3-D maps of the tobacco mosaic virus in under two days. By comparison, this would have taken several months of work just a few years ago and perhaps several weeks using conventional methods today.

"We can now go from inserting the virus into the microscope to having a 3-D map in 24 hours," says Milligan, adding that the fear of failure should no longer be a limiting factor for experiments.

Still, the automation is not fully implemented, so one of the immediate goals of the Automated Molecular Imaging Group is to see their software used for practical applications, something that their coming to TSRI will facilitate.

"At the moment we need to make the technique very efficient and very general, and get it out to the community," says Carragher. "We can do it, and now we want to be able to do it routinely for anybody."

Additional plans include the design of technology that would make EM high-throughput. This includes a robotic specimen handler that Carragher and Potter have been experimenting with that would allow the instruments to be left alone to collect and analyze even larger sets of data.

At present, several people control the microscopes and discuss the images as they collect data. This is what Milligan and the others envisioned when the original plans were drawn. They also anticipated people peering through the glass wall of the control room.

"It's a very easy way to communicate what we are doing," Carragher reports.