Riboswitches are structurally dynamic RNA molecules, undergoing changes in shape as they perform their regulatory functions. Riboswitches typically have two domains: a ligand-binding “aptamer” domain that changes conformation when it binds to a specific ligand, which then sends a signal to an “expression platform” domain that regulates use of the RNA.

Thus far, structures have been obtained for the aptamer domains, but the motions caused by ligand binding have been tricky to observe by crystallography: crystals typically freeze molecules in one shape, so crystallographic structures of unbound aptamers typically look very similar to the ligand-bound form. Researchers are using XFEL radiation sources in a clever way to help surmount this limitation and observe directly what happens when a ligand binds.

Free Electron Lasers

Free electron lasers (FEL) provide very bright, but very short, pulses of X-rays. In XFEL experiments, a stream of very tiny crystals is flowed past the beam, and each time a crystal is caught in one of the pulses, it creates an instantaneous diffraction pattern that captures the molecules in the crystal at a defined moment of time. By collecting similar diffraction patterns from many crystals caught in random orientations by the X-ray beam, researchers build up a full diffraction data set.


To capture the dynamics of molecules, researchers start a reaction and then collect diffraction patterns at a given time after the reaction starts. In studies of photoactive yellow protein, researchers flash the crystals with light and then watch what happens. For example, in the case of riboswitches, researchers add the specific signaling molecule, in this case, adenine. Two structures are shown here. The first shows the riboswitch without adenine (PDB entry 5e54). For the second, researchers mixed the crystals with adenine, waited 10 minutes, then gathered XFEL data. The adenine-bound form shows a large change in conformation that creates a stable double helix at the end of the riboswitch.

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