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We report an experimental demonstration of real-time in situ x-ray diffraction investigations of clustering and dynamic strain in early stages of nanoparticle growth in Al–Cu alloys. Simulations involving a simplified model of local strain are well correlated with the x-ray diffraction data, suggesting a redistribution of point defects and the formation of nanoscale clusters in the bulk material. A modal, representative nanoparticle size is determined subsequent to the final stage of artificial aging. Such investigations are imperative for the understanding, and ultimately the control, of nanoparticle nucleation and growth in this technologically important alloy. [ABSTRACT FROM AUTHOR]

• MicroED and SFX are capable of determining structures of proteins from nano/microcrystals. • The MicroED and SFX techniques are highly complementary. • SFX provides significant advantages for time-resolved studies. • MicroED produces structures from a very small number of microcrystals. In recent years, nano and microcrystals have emerged as a valuable source of high-resolution structural information owing to the invention of serial femtosecond crystallography (SFX) with X-ray free electron lasers and microcrystal electron diffraction (MicroED) using electron cryomicroscopes. Once considered useless for structure determination, nano/microcrystals now confer significant advantages for static and time-resolved structure determination from a wide variety of difficult-to-study targets. MicroED has been used to obtain sub-Ångstrom resolution maps in which hydrogen atoms can be clearly resolved from only a few nano/microcrystals, while SFX has been used to probe protein dynamics following reaction initiation on time scales from femtoseconds to minutes. We review these two complementary techniques and their abilities for high-resolution structure determination. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe-C-O angle of ~142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry. [ABSTRACT FROM AUTHOR]

Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics. [ABSTRACT FROM AUTHOR]

The use of coherent X-ray lasers for structural biology allows the use of nanometre diameter X-ray beams with large beam divergence. Their application to the structure analysis of protein nanocrystals and single particles raises new challenges and opportunities. We discuss the form of these coherent convergent-beam (CCB) hard X-ray diffraction patterns and their potential use for time-resolved crystallography, normally achieved by Laue (polychromatic) diffraction, for which the monochromatic laser radiation of a free-electron X-ray laser is unsuitable. We discuss the possibility of obtaining single-shot, angle-integrated rocking curves from CCB patterns, and the dependence of the resulting patterns on the focused beam coordinate when the beam diameter is larger or smaller than a nanocrystal, or smaller than one unit cell. We show how structure factor phase information is provided at overlapping interfering orders and how a common phase origin between different shots may be obtained. Their use in refinement of the phase-sensitive intensity between overlapping orders is suggested. [ABSTRACT FROM AUTHOR]

The potential for nondestructive in situ detection of the formation of weakly diffracting nanoparticles has been confirmed by a combination of experiment and simulation. A triple axis diffractometer was used to collect two-dimensional reciprocal space maps of diffracted synchrotron x-rays from nanoscale Al2Cu precipitates embedded in a bulk metallic matrix. The appearance and asymmetric profile of the monochromator pseudostreaks are demonstrated to be indicative of the sensitivity of the technique to both the presence and orientation of the nanoparticles. This is a fundamental step toward in situ detection of sparsely dispersed, embedded nanoparticles and to quantitative temporal studies of particle number, scale, and dispersion. [ABSTRACT FROM AUTHOR]

DatView is a new graphical user interface (GUI) for plotting parameters to explore correlations, identify outliers and export subsets of data. It was designed to simplify and expedite analysis of very large unmerged serial femtosecond crystallography (SFX) data sets composed of indexing results from hundreds of thousands of microcrystal diffraction patterns. However, DatView works with any tabulated data, offering its functionality to many applications outside serial crystallography. In DatView's user‐friendly GUI, selections are drawn onto plots and synchronized across all other plots, so correlations between multiple parameters in large multi‐parameter data sets can be rapidly identified. It also includes an item viewer for displaying images in the current selection alongside the associated metadata. For serial crystallography data processed by indexamajig from CrystFEL [White, Kirian, Martin, Aquila, Nass, Barty & Chapman (2012). J. Appl. Cryst.45, 335–341], DatView generates a table of parameters and metadata from stream files and, optionally, the associated HDF5 files. By combining the functionality of several commonly needed tools for SFX in a single GUI that operates on tabulated data, the time needed to load and calculate statistics from large data sets is reduced. This paper describes how DatView facilitates (i) efficient feedback during data collection by examining trends in time, sample position or any parameter, (ii) determination of optimal indexing and integration parameters via the comparison mode, (iii) identification of systematic errors in unmerged SFX data sets, and (iv) sorting and highly flexible data filtering (plot selections, Boolean filters and more), including direct export of subset CrystFEL stream files for further processing. [ABSTRACT FROM AUTHOR]

The article discusses the capture of high-resolution intermediates of photoactive yellow protein (PYP), a bacterial blue light photoreceptor, with time-resolved serial femtosecond crystallography (TR-SFX), which uses ultrashort pulses from x-ray free electron lasers (XFELs). Topics include the challenges of TR-SFX, TR-SFX's advantages over synchrotron-based, time-resolved Laue crystallography, and difference electron density (DED) maps created by using microcrystals of PYP as a model system.

The increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 µm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 µm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures. [ABSTRACT FROM AUTHOR]

μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448–605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP‐μNS(448–605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP‐μNS(448–605) baculovirus formed rod‐shaped microcrystals (5–15 µm in length) which were reconstituted in high‐viscosity media (LCP and agarose) and investigated by serial femtosecond X‐ray diffraction using viscous jets at an X‐ray free‐electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit‐cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron‐density maps were obtained. While more diffraction data are required to solve the structure of μNS(448–605), the current experimental strategy, which couples high‐viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein. [ABSTRACT FROM AUTHOR]

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X‐ray free‐electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X‐ray beam using a liquid jet injector. Owing to the pulsed nature of the X‐ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two‐photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long‐term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal‐laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly. [ABSTRACT FROM AUTHOR]

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5Å ) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods. [ABSTRACT FROM AUTHOR]

Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å , thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump-probe experiments at subpicosecond time resolution. [ABSTRACT FROM AUTHOR]

X-ray crystallography of G protein--coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. [ABSTRACT FROM AUTHOR]

A processing pipeline for diffraction data acquired using the `serial crystallography' methodology with a free-electron laser source is described with reference to the crystallographic analysis suite CrystFEL and the pre-processing program Cheetah. A detailed analysis of the nature and impact of indexing ambiguities is presented. Simulations of the Monte Carlo integration scheme, which accounts for the partially recorded nature of the diffraction intensities, are presented and show that the integration of partial reflections could be made to converge more quickly if the bandwidth of the X-rays were to be increased by a small amount or if a slight convergence angle were introduced into the incident beam. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia. • X-ray free-electron laser unveils alternative protein structure of lipoprotein • Advanced molecular dynamics explore conformational space among solved structures • Virtual ligand screening shows possible lead fragments for potential drug therapies Using X-ray free-electron laser (XFEL) technology, Zook et al. describe an alternative protein conformation of the virulence determinant Flpp3 and use molecular dynamics (MD) to investigate conformational landscapes between these conformations. MD identifies several intermediate conformations that allow these conformational transitions to occur at physiologically relevant temperatures and provide insight to potential future drug therapies for the potentially deadly disease tularemia. [ABSTRACT FROM AUTHOR]