A method for recovering complex structure factors from many simultaneously excited Bragg beam in- tensities is described. The method is applied to simulated transmission electron diffraction data over a wide range of crystal thickness and beam energies. The method is based on iterated projections between structure and scattering matrices, which are related by a matrix unit ary transformation, exponential, which we invert. The algorithm removes multiple-scattering perturbations from diffraction data and might be extended to other fields, including X-ray and neutron diffraction and cryo-electron microscopy. Because coherent multiple scattering involves interference between Bragg beams, the method also solves the phase problem. Unlike dynamical inversion from electron microscope images or ptychography data, the method, which starts with Bragg beam intensities, provides complex structure factors unaffected by focusing errors or resolution limitations imposed by lenses. We provide inversions from simulated data with 441 simultaneously excited Bragg beams over a range of thickness and beam energy. We discuss the retrieval of chirality information from enantiomorphs, the efficient incorporation of symmetry information using the irreducible representation of the group of structure matrices, and the effect of HOLZ lines to provide three-dimensional information.

An iterated projection algorithm (N-Phaser) is developed that reconstructs a scattering potential from N-beam multiple Bragg scattered intensities. The method may be used to eliminate multiple scattering artifacts from electron diffraction data, solving the phase problem and increasing the thicknesses of samples used in materials science, solid-state chemistry, and small molecule crystallography. For high-energy transmission electron diffraction, we show that the algorithm recovers accurate complex structure factors from a wide range of thicknesses, orientations, and relativistic beam energies, and does not require known thickness or atomic-resolution data if sufficient multiple scattering occurs. Extensions to Cryo-electron microscopy and Micro-electron diffraction are suggested.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl^- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.

A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales 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 photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.

Background

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases.

Results

Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s.

Conclusions

MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.