Li and his collaborators studied a phytochrome from a common bacterium that is quite similar in biochemistry and function to those found in plants, but easier to isolate. Plant biologist Richard Vierstra of the University of Wisconsin provided the purified samples.
At Brookhaven, Li's group used two imaging techniques. First, they applied a layer of heavy metal dye to the purified phytochrome molecules to make them more visible, and viewed them using an electron microscope. This produced many two-dimensional images from a variety of angles to give the researchers a rough outline of the phytochrome map.
The scientists also froze the molecules in solution to produce another set of images that would be free of artifacts from the staining technique. For this set of images, the scientists used a cryo-electron microscope.
Using computers to average the data from each technique and then combine the information, the scientists were able to construct a three-dimensional map of the full phytochrome structure. The scientists then fitted the previously determined detailed structures of phytochrome fragments into their newly derived 3-D map to build an atomic model for the whole phytochrome.
Though the scientists knew the phytochrome was composed of two "sister" units, forming a dimer, the new structure revealed a surprisingly long twisted area of contact between the two individual units, with a good deal of flexibility at the untwisted ends. The structure supports the idea that the absorption of light somehow adjusts the strength or orientation of the contact, and through a series of conformation changes, transmits a signal down the length of the molecular interface. The scientists confirmed the proposed structural changes during photo-conversion by mutagenesis and biochemical assay.
The scientists studie
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DOE/Brookhaven National Laboratory