Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

Light-driven sodium pumps actively transport small cations across cellular membranes 1 .They are used by microbes to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. While resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved 2,3 , it is unclear how structural alterations over time allow sodium translocation against a concentration gradient. Using the Swiss X-ray Free Electron Laser 4 , we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. Highresolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data in combination with quantum chemical calculations indicate transient binding of a sodium ion close to the retinal within one millisecond. In the last structural intermediate at 20 ms after activation, we identified a potential second sodium binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.

Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid–96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

We show that the combination of X-ray scattering with a nanofocused beam and X-ray cross correlation analysis is an efficient means for the full structural characterization of mesocrystalline nanoparticle assemblies with a single experiment. We analyze several hundred diffraction patterns of individual sample locations, i.e. individual grains, to obtain a meaningful statistical distribution of the superlattice and atomic lattice ordering. Simultaneous small-and wide-angle X-ray scattering of the same sample location allows us to determine the structure and orientation of the superlattice as well as the angular correlation of the first two Bragg peaks of the atomic lattices, their orientation with respect to the superlattice, and the average orientational misfit due to local structural disorder. This experiment is particularly advantageous for synthetic mesocrystals made by the simultaneous self-assembly of colloidal nanocrystals and surfacefunctionalization with conductive ligands. While the structural characterization of such materials has been challenging so far, the present method now allows correlating mesocrystalline structure with optoelectronic properties. Mesocrystals (MC) are three-dimensional arrays of iso-oriented single-crystalline particles with an individual size between 1 -1000 nm. [1][2][3][4][5] Their physical properties are largely determined by structural coherence, for which the angular correlation between their individual atomic lattices and the underlying superlattice of nanocrystals (NC) is a key ingredient. 1,2 Colloidal NCs stabilized by organic surfactants have been shown to pose excellent building blocks for the design of synthetic MCs with tailored structural properties which are conveniently obtained by self-assembly of NCs from solution on a solid or liquid substrate by exploiting ligand-ligand 3 interactions. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] Typically, the utilized ligands consist of wide-gap, bulky hydrocarbons which render the MCs insulating. [26][27][28][29][30][31][32][33] MCs obtained in this way exhibit average grain sizes of ~150 µm 2 , which enables a detailed characterization by electron and/or X-ray microscopy. 34 Since the optoelectronic properties of PbS NC ensembles bear many opportunities for applications in solar cells or photodetectors, a number of ligand exchange procedures with small organic or inorganic molecules as well as single atom passivation strategies have been developed, all of which greatly increase the carrier mobilities within the SL of NCs. 28,33,[35][36][37][38][39][40][41][42][43][44] Due to the short interparticle spacing imposed by these ligands, structural coherence is mostly lost in such superlattices, but in rare cases it has been demonstrated that significant long-range order and even mesocrystallinity can be preserved. 25,35,45 However, a persisting problem of these protocols is that they are prone to introduce defects in the superlattice structure with some degree of granularity and signifi...