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.

Resampling, full occupancy structural refinement and analysis were performed by V.A.G, G.K. and A.V. Integration within a sphere and statistical tests were performed by C.W., R.N. and P.Bå.. SVD analysis was performed by A.V. Quantum Mechanics/Molecular Mechanics analysis were performed by D.M., H.L.L and G.G.. Time-resolved IR spectroscopy measurements were performed by J.K, M.M and S.W. The manuscript was prepared by R.N.,

The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RC). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RC serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.

X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.

A major goal for X‐ray free‐electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one‐dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F‐actin, and amyloid fibrils), which when intersected by femtosecond X‐ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F‐actin can be flow‐aligned to a disorientation of approximately 5 degrees. Using this XFEL‐based technique, we determine that gelsolin amyloids are comprised of stacked β‐strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α‐synuclein amyloids.

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The X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium.