Serial femtosecond crystallography using ultrashort pulses from X-ray Free Electron Lasers (XFELs) offers the possibility to study light-triggered dynamics of biomolecules. Using microcrystals of the blue light photoreceptor, photoactive yellow protein, as a model system, we present high resolution, time-resolved difference electron density maps of excellent quality with strong features, which allow the determination of structures of reaction intermediates to 1.6 Å resolution. These results open the way to the study of reversible and non-reversible biological reactions on time scales as short as femtoseconds under conditions which maximize the extent of reaction initiation throughout the crystal.

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A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes following photon absorption. The initial step is often the photo-isomerization of a conjugated chromophore. Isomerization occurs on ultrafast timescales, and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans to cis isomerization of the chromophore in photoactive yellow protein. Femtosecond, hard X-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on PYP microcrystals over the time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.

Scaling the Si MOSFET is revisited. Requirements on subthreshold leakage control force conventional scaling to use high doping as the device dimension penetrates into the deep-submicrometer regime, leading to undesirable large junction capacitance and degraded mobility. By studying the scaling of fully depleted SO1 devices, we note the important concept of controlling horizontal leakage through vertical structures. Several structural variations of conventional SO1 structures are discussed in terms of a natural length scale to guide the design. The concept of Vertical Doping Engineering can also be realized in bulk Si to obtain good subthreshold characteristics without large junction capacitance or heavy channel doping. 'Monte Carlo simulations are required to predict the device performance in the turn-on regime [ 5 ] .

There are many instances when the structure of a weakly-scattering spinning object in flight must be determined to high resolution. Examples range from comets to nanoparticles and single molecules. The latter two instances are the subject of intense current interest. Substantial progress has recently been made in illuminating spinning single particles in flight with powerful X-ray bursts to determine their structure 1,2 , with the ultimate goal of determining the structure of single molecules 3,4,5,6,7 . However, proposals to reconstruct the molecular structure from diffraction "snapshots" of unknown orientation require ~1000x more signal than available from next-generation sources 8 . Using a new approach, we demonstrate the recovery of the structure of a weakly scattering macromolecule at the anticipated next-generation X-ray source intensities. Our work closes a critical gap in determining the structure of single molecules and nanoparticles by X-ray methods, and opens the way to reconstructing the structure of spinning, or randomly-oriented objects at extremely low signal levels. Other potential applications include low-dose electron microscopy, ultra-low-signal tomography of non-stationary objects without orientational information, and the study of heavenly bodies.

A Brownian machine, a tiny device buffeted by the random motions of molecules in the environment, is capable of exploiting these thermal motions for many of the conformational changes in its work cycle. Such machines are now thought to be ubiquitous, with the ribosome, a molecular machine responsible for protein synthesis, increasingly regarded as prototypical. Here we present a new analytical approach capable of determining the free-energy landscape and the continuous trajectories of molecular machines from a large number of snapshots obtained by cryogenic electron microscopy. We demonstrate this approach in the context of experimental cryogenic electron microscope images of a large ensemble of nontranslating ribosomes purified from yeast cells. The freeenergy landscape is seen to contain a closed path of low energy, along which the ribosome exhibits conformational changes known to be associated with the elongation cycle. Our approach allows model-free quantitative analysis of the degrees of freedom and the energy landscape underlying continuous conformational changes in nanomachines, including those important for biological function.cryo-electron microscopy | elongation cycle | manifold embedding | nanomachines | translation

Cryo-electron microscopy, when combined with single-particle reconstruction, is a powerful method for studying macromolecular structure. Recent developments in detector technology have pushed the resolution into a range comparable to that of X-ray crystallography. However, cryo-EM is able to separate and thus recover the structure of each of several discrete structures present in the sample. For the more general case involving continuous structural changes, a novel technique employing manifold embedding has been recently demonstrated. Potentially, the entire work-cycle of a molecular machine may be observed as it passes through a continuum of states, and its freeenergy landscape may be mapped out. This technique will be outlined and discussed in the context of its application to a large single-particle dataset of yeast ribosomes.

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