Riboswitches is RNA structural elements generally located in the 5ˈuntranslated region (3ˈUTR) of mRNA. In the genetic regulation, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis for this mechanism requires the ability to study structural changes over time. We apply femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction prior to diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction involving two apo, one ligand-bound intermediate, and the final bound states. These structures support a reaction mechanism model with at least four states and illustrate the structural basis for signal transmission. The two apo conformers differ significantly in the three-way junction and the P1 switch helix relative to the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With a >10-minute delay, the RNA molecules were fully converted to the bound state, in which the substantial conformational changes resulted in conversion of the space group. Such drastic changes in crystallo highlight the important opportunities that micro/nanocrystals may offer in these and similar time-resolved diffraction studies. These results all together demonstrate the potential of "mix-and-inject" time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those involving large conformational changes.
Fresnel edge fringes observed in a lensless point projection field-emission electron microscope operating at 90 eV have been studied and found to be formally equivalent to the fringes observed in transmission electron microscopy (TEM) under weak scattering conditions at the edge of an opaque object. The tip-to-spectrum distance z1 plays the role of the objective lens defocus setting Δf in conventional TEM. The image magnification, effective source size, transverse coherence width, instrumental resolution, and source brightness are all obtained from an analysis of the fringe spacings and intensity. The quantum mechanical upper limit on source brightness, as well as relationships among beam brightness, coherence parameters, and degeneracy, are discussed, and the degeneracy measured from experimental Fresnel fringes.
A new crysallographic technique has been developed, which has been applied to the problem of locating the cations in a natural olivine crystal with the composition (Mg(0.90)Fe(0.10)Ni(0.004)Mn(0.002))(2)SiO(4). The method uses the variation of characteristic x-ray emission with the direction of an exciting electron beam in an analytical transmission electron microscope. It may be applied to nanometer-sized areas and to concentrations as low as 0.1 atomic percent, is capable of distinguishing neighbors in the periodic table, and does not require external standards. The iron atoms in this crystal are evenly distributed between the two available crystal sites M1 and M2 (49.6 +/- 1 percent on M1), whereas the trace elements nickel and manganese occupy the M1 and M2 positions, respectively (97 +/- 5 percent nickel on M1 and 1 +/- 5 percent manganese on M1).
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