Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

The making and breaking of atomic bonds are essential processes in chemical reactions. Although the ultrafast dynamics of bond breaking have been studied intensively using time-resolved techniques, it is very difficult to study the structural dynamics of bond making, mainly because of its bimolecular nature. It is especially difficult to initiate and follow diffusion-limited bond formation in solution with ultrahigh time resolution. Here we use femtosecond time-resolved X-ray solution scattering to visualize the formation of a gold trimer complex, [Au(CN)2(-)]3 in real time without the limitation imposed by slow diffusion. This photoexcited gold trimer, which has weakly bound gold atoms in the ground state, undergoes a sequence of structural changes, and our experiments probe the dynamics of individual reaction steps, including covalent bond formation, the bent-to-linear transition, bond contraction and tetramer formation with a time resolution of ∼500 femtoseconds. We also determined the three-dimensional structures of reaction intermediates with sub-ångström spatial resolution. This work demonstrates that it is possible to track in detail and in real time the structural changes that occur during a chemical reaction in solution using X-ray free-electron lasers and advanced analysis of time-resolved solution scattering data.

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

We present the first experimental demonstration of lensless diffractive imaging using coherent soft x rays generated by a tabletop soft-x-ray source. A 29 nm high harmonic beam illuminates an object, and the subsequent diffraction is collected on an x-ray CCD camera. High dynamic range diffraction patterns are obtained by taking multiple exposures while blocking small-angle diffraction using beam blocks of varying size. These patterns reconstruct to images with 214 nm resolution. This work demonstrates a practical tabletop lensless microscope that promises to find applications in materials science, nanoscience, and biology.