Access: Piecing it Together

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To create the models, also known as reconstructions, the team uses a technique known as single-particle analysis. Particles are first selected from the microscopic images, or micrographs. A single micrograph can contain hundreds of thousands of particles with random orientations, so the particles are first grouped by similar orientation. The particles within a group are then aligned to each other and averaged together. These groups represent views of the particle that are a few degrees different from one another.

From this collection of views, a preliminary three-dimensional model is built. At this point, determining the symmetry of the particle is critical. Although symmetry is not generally known, the ways in which sections of the particle mirror one another along a given axis have a tremendous impact on the computation and refinement of the final model.

Reconstruction of a herpes simplex virus capsid at 8.5 angstrom.

"Often the biochemistry of the particle will give you at least a hint, and generally you can get an idea based on the appearance of individual projections at a given orientation," says Ludtke. But there are also times that researchers rely on statistical techniques. "We go through a several-stage process, running refinements with several different possible symmetries to try to discover the correct answer."

Once an initial model is created, the model is refined. A version is compared to the raw data—the projections of the particle at different orientations—and adjusted. In the most computationally taxing aspect of the process, this loop continues until the data and the model match. The 20-angstrom model of alpha-crystallin, for example, required 10 refinement loops. Each loop of the model, which had no symmetry and was thus more difficult than many, took 48 hours on an eight-processor Origin2000 configuration. To get that model down to 15 angstroms, the computational requirements will go up by at least a factor of ten.