Few-fs resolution of a photoactive protein traversing a conical intersection.

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate potential energy surfaces. Posited almost a century ago1, conical intersections remain the subject of intense scientific interest2-5. In biology, they have a pivotal role in vision, photosynthesis and DNA stability6. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around ten atoms, on timescales of about 100 fs or longer7. Spectroscopy can achieve better time resolutions8, but provides indirect structural information. Here we present few-femtosecond, atomic-resolution videos of photoactive yellow protein, a 2,000-atom protein, passing through a conical intersection. These videos, extracted from experimental data by machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process and elucidate the topography of the electronic potential energy surfaces involved.

Single-particle structure determination by X-ray free-electron lasers: Possibilities and challenges.

Single-particle structure recovery without crystals or radiation damage is a revolutionary possibility offered by X-ray free-electron lasers, but it involves formidable experimental and data-analytical challenges. Many of these difficulties were encountered during the development of cryogenic electron microscopy of biological systems. Electron microscopy of biological entities has now reached a spatial resolution of about 0.3 nm, with a rapidly emerging capability to map discrete and continuous conformational changes and the energy landscapes of biomolecular machines. Nonetheless, single-particle imaging by X-ray free-electron lasers remains important for a range of applications, including the study of large "electron-opaque" objects and time-resolved examination of key biological processes at physiological temperatures. After summarizing the state of the art in the study of structure and conformations by cryogenic electron microscopy, we identify the primary opportunities and challenges facing X-ray-based single-particle approaches, and possible means for circumventing them.

High-resolution structure of viruses from random diffraction snapshots.

The advent of the X-ray free-electron laser (XFEL) has made it possible to record diffraction snapshots of biological entities injected into the X-ray beam before the onset of radiation damage. Algorithmic means must then be used to determine the snapshot orientations and thence the three-dimensional structure of the object. Existing Bayesian approaches are limited in reconstruction resolution typically to 1/10 of the object diameter, with the computational expense increasing as the eighth power of the ratio of diameter to resolution. We present an approach capable of exploiting object symmetries to recover three-dimensional structure to high resolution, and thus reconstruct the structure of the satellite tobacco necrosis virus to atomic level. Our approach offers the highest reconstruction resolution for XFEL snapshots to date and provides a potentially powerful alternative route for analysis of data from crystalline and nano-crystalline objects.