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We present the first lasing results of SwissFEL, a hard X-ray free-electron laser (FEL) that recently came into operation at the Paul Scherrer Institute in Switzerland. SwissFEL is a very stable, compact and cost-effective X-ray FEL facility driven by a low-energy and ultra-low-emittance electron beam travelling through short-period undulators. It delivers stable hard X-ray FEL radiation at 1-A wavelength with pulse energies of more than 500 μJ, pulse durations of ~30 fs (root mean square) and spectral bandwidth below the per-mil level. Using special configurations, we have produced pulses shorter than 1 fs and, in a different set-up, broadband radiation with an unprecedented bandwidth of ~2%. The extremely small emittance demonstrated at SwissFEL paves the way for even more compact and affordable hard X-ray FELs, potentially boosting the number of facilities worldwide and thereby expanding the population of the scientific community that has access to X-ray FEL radiation. The first lasing results at SwissFEL, an X-ray free-electron laser, are presented, highlighting the facility’s unique capabilities. A general comparison to other major facilities is also provided.

Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets., IUCrJ, 7 (6), ISSN:2052-2525

Use of the charge-integrating JUNGFRAU detector for fluorescence detection is investigated, and its applications in macromolecular crystallography at synchrotron and X-ray free-electron laser sources are demonstrated., Detection of heavy elements, such as metals, in macromolecular crystallography (MX) samples by X-ray fluorescence is a function traditionally covered at synchrotron MX beamlines by silicon drift detectors, which cannot be used at X-ray free-electron lasers because of the very short duration of the X-ray pulses. Here it is shown that the hybrid pixel charge-integrating detector JUNGFRAU can fulfill this function when operating in a low-flux regime. The feasibility of precise position determination of micrometre-sized metal marks is also demonstrated, to be used as fiducials for offline prelocation in serial crystallography experiments, based on the specific fluorescence signal measured with JUNGFRAU, both at the synchrotron and at SwissFEL. Finally, the measurement of elemental absorption edges at a synchrotron beamline using JUNGFRAU is also demonstrated.

In the ciliate Euplotes raikovi, water-borne protein pheromones promote the vegetative cell growth and mating by competitively binding as autocrine and heterologous signals to putative cell receptors represented by membrane-bound pheromone isoforms. A previously determined crystal structure of pheromone Er-1 supported a pheromone/receptor binding model in which strong protein-protein interactions result from the cooperative utilization of two distinct types of contact interfaces that arrange molecules into linear chains, and these into two-dimensional layers. We have now determined the crystal structure of a new pheromone, Er-13, isolated from cultures that are strongly mating reactive with cultures source of pheromone Er-1. The comparison between the Er-1 and Er-13 crystal structures reinforces the fundamental of the cooperative model of pheromone/receptor binding, in that the molecules arrange into linear chains taking a rigorously alternate opposite orientation reflecting the presumed mutual orientation of pheromone and receptor molecules on the cell surface. In addition, the comparison provides two new lines of evidence for a univocal rationalization of observations on the different behaviour between the autocrine and heterologous pheromone/receptor complexes. (i) In the Er-13 crystal, chains do not form layers which thus appear to be an over-structure unique to the Er-1 crystal, not essential for the pheromone signalling mechanisms. (ii) In both crystal structures, the intra-chain interfaces are equally derived from burying amino-acid side-chains mostly residing on helix-3 of the three-helical pheromone fold. This helix is thus identified as the key structural motif underlying the pheromone activity, in line with its tight intra- and interspecific structural conservation.

X-ray free-electron lasers (FELs) deliver ultrabright X-ray pulses, but not the sequences of phase-coherent pulses required for time-domain interferometry and control of quantum states. For conventional split-and-delay schemes to produce such sequences, the challenge stems from extreme stability requirements when splitting Ångstrom wavelength beams, where the tiniest path-length differences introduce phase jitter. We describe an FEL mode based on selective electron-bunch degradation and transverse beam shaping in the accelerator, combined with a self-seeded photon emission scheme. Instead of splitting the photon pulses after their generation by the FEL, we split the electron bunch in the accelerator, prior to photon generation, to obtain phase-locked X-ray pulses with subfemtosecond duration. Time-domain interferometry becomes possible, enabling the concomitant program of classical and quantum optics experiments with X-rays. The scheme leads to scientific benefits of cutting-edge FELs with attosecond and/or high-repetition rate capabilities, ranging from the X-ray analog of Fourier transform infrared spectroscopy to damage-free measurements., Proceedings of the National Academy of Sciences of the United States of America, 119 (7), ISSN:0027-8424, ISSN:1091-6490

International audience; The Bernina instrument at the SwissFEL Aramis hard X-ray free-electron laser is designed for studying ultrafast phenomena in condensed matter and material science. Ultrashort pulses from an optical laser system covering a large wavelength range can be used to generate specific non-equilibrium states, whose subsequent temporal evolution can be probed by selective X-ray scattering techniques in the range 2–12 keV. For that purpose, the X-ray beamline is equipped with optical elements which tailor the X-ray beam size and energy, as well as with pulse-to-pulse diagnostics that monitor the X-ray pulse intensity, position, as well as its spectral and temporal properties. The experiments can be performed using multiple interchangeable endstations differing in specialization, diffractometer and X-ray analyser configuration and load capacity for specialized sample environment. After testing the instrument in a series of pilot experiments in 2018, regular user operation begins in 2019.

Three-dimensional intensities were reconstructed from serial crystallography data using two-dimensional crystals., Serial femtosecond crystallography of two-dimensional membrane-protein crystals at X-ray free-electron lasers has the potential to address the dynamics of functionally relevant large-scale motions, which can be sterically hindered in three-dimensional crystals and suppressed in cryocooled samples. In previous work, diffraction data limited to a two-dimensional reciprocal-space slice were evaluated and it was demonstrated that the low intensity of the diffraction signal can be overcome by collecting highly redundant data, thus enhancing the achievable resolution. Here, the application of a newly developed method to analyze diffraction data covering three reciprocal-space dimensions, extracting the reciprocal-space map of the structure-factor amplitudes, is presented. Despite the low resolution and completeness of the data set, it is shown by molecular replacement that the reconstructed amplitudes carry meaningful structural information. Therefore, it appears that these intrinsic limitations in resolution and completeness from two-dimensional crystal diffraction may be overcome by collecting highly redundant data along the three reciprocal-space axes, thus allowing the measurement of large-scale dynamics in pump–probe experiments.

Optical-domain Transient Grating (TG) spectroscopy is a versatile background-free four-wave-mixing technique used to probe vibrational, magnetic and electronic degrees of freedom in the time domain. The newly developed coherent X-ray Free Electron Laser sources allow its extension to the X-ray regime. Xrays offer multiple advantages for TG: their large penetration depth allows probing the bulk properties of materials, their element-specificity can address core-excited states, and their short wavelengths create excitation gratings with unprecedented momentum transfer and spatial resolution. We demonstrate for the first time TG excitation in the hard X-ray range at 7.1 keV. In Bismuth Germanate (BGO), the nonresonant TG excitation generates coherent optical phonons detected as a function of time by diffraction of an optical probe pulse. This experiment demonstrates the ability to probe bulk properties of materials and paves the way for ultrafast coherent four-wave-mixing techniques using X-ray probes and involving nanoscale TG spatial periods., Comment: 11 pages, 4 figures

The emergence of new high brilliance and high coherence facilities such as X-ray Free Electron Lasers (XFELs) and 4th generation synchrotrons open a new era in X-ray optics. Dynamical diffraction effects before disregarded are starting to play a role in the beam control of large scale facilities. In the case of XFEL facilities the temporal characteristics of the dynamical diffraction by thin perfect crystals can be used as a tool to generate femtosecond monochromatic pulses, in the case of self-seeding in the hard X-ray regime, but could even be used as method to characterize materials in this temporal range. In this contribution we present the first steps in the understanding of the spatial-displacement dependence of forward beams diffracted by thin crystals. The data collected by this technique is compared with crystal models based in dynamical diffraction theory. This type of study could open a new field to understand low strain materials in the femtosecond regime.

A microcrystal-prelocation method is demonstrated using low-dose 2D full-field propagation-based X-ray phase-contrast imaging on samples with an essentially flat geometry for automated serial crystallography data collection at a microfocus macromolecular crystallography beamline., Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

The first experimental observation of transverse spatial echoes generated by forward Bragg diffraction of an X-ray beam propagating through a perfect thin crystal is reported., Time-delayed, narrow-band echoes generated by forward Bragg diffraction of an X-ray pulse by a perfect thin crystal are exploited for self-seeding at hard X-ray free-electron lasers. Theoretical predictions indicate that the retardation is strictly correlated to a transverse displacement of the echo pulses. This article reports the first experimental observation of the displaced echoes. The displacements are in good agreement with simulations relying on the dynamical diffraction theory. The echo signals are characteristic for a given Bragg reflection, the structure factor and the probed interplane distance. The reported results pave the way to exploiting the signals as an online diagnostic tool for hard X-ray free-electron laser seeding and for dynamical diffraction investigations of strain at the femtosecond timescale.

IUCrJ 5(1), 103 - 117 (2018). doi:10.1107/S2052252517017043, Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals., Published by IUCr, Chester

The extension of transient grating spectroscopy to the x-ray regime will create numerous opportunities, ranging from the study of thermal transport in the ballistic regime to charge, spin, and energy transfer processes with atomic spatial and femtosecond temporal resolution. Studies involving complicated split-and-delay lines have not yet been successful in achieving this goal. Here we propose a novel, simple method based on the Talbot effect for converging beams, which can easily be implemented at current x-ray free electron lasers. We validate our proposal by analyzing printed interference patterns on polymethyl methacrylate and gold samples using similar to 3 keV X-ray pulses. (C) 2019 Optical Society of America

Among protists, pheromones have been identified in a great variety of algal species for their activity in driving gamete-gamete interactions for fertilization. Analogously in ciliates, pheromones have been identified for their activity in inducing the sexual phenomenon of conjugation. Although this identification was pioneered by Kimball more than fifty years ago, an effective isolation and chemical characterization of ciliate pheromones has remained confined to species of Blepharisma, Dileptus and Euplotes. In Euplotes species, in which the molecular structures have been determined, pheromones form species-specific families of structurally homologous helical, cysteine-rich, highly-stable proteins. Being structurally homologous, they can bind cells in competition with one another, raising interesting functional analogies with the families of growth factors and cytokines that regulate cell differentiation and development in higher organisms. In addition to inducing conjugation by binding cells in heterologous fashion, Euplotes pheromones act also as autocrine growth factors by binding to, and promoting the vegetative reproduction of the same cells from which they originate. This autocrine activity is most likely primary, providing a concrete example of how the original function of a molecule can be obscured during evolution by the acquisition of a new one.

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

Next generation X ray sources based on the X ray Free Electron Laser (XFEL) concept will provide highly coherent ultrashort pulses of soft and hard X rays with peak intensity many orders of magnitude higher than that of a synchrotron. These pulses will allow studies of femtosecond dynamics at nanometer resolution and with chemical selectivity. They will produce diffraction images of organic and inorganic nanostructures without deleterious effects of radiation damage. © Schweizerische Chemische Gesellschaft.

The SwissFEL X-ray Free Electron Laser (XFEL) facility started construction at the Paul Scherrer Institute (Villigen, Switzerland) in 2013 and will be ready to accept its first users in 2018 on the Aramis hard X-ray branch. In the following sections we will summarize the various aspects of the project, including the design of the soft and hard X-ray branches of the accelerator, the results of SwissFEL performance simulations, details of the photon beamlines and experimental stations, and our first commissioning results.

X-ray techniques have long been applied to chemical research, ranging from powder diffraction tools to analyse material structure to X-ray fluorescence measurements for sample composition. The development of high-brightness, accelerator-based X-ray sources has allowed chemists to use similar techniques but on more demanding samples and using more photon-hungry methods. X-ray Free Electron Lasers (XFELs) are the latest in the development of these large-scale user facilities, opening up new avenues of research and the possibility of more advanced applications for a range of research. The SwissFEL XFEL project at the Paul Scherrer Institute will begin user operation in the hard X-ray (2.1–12.4 keV) photon energy range in 2018 with soft X-ray (240–1930 eV) user operation to follow and here we will present the details of this project, it's operating capabilities, and some aspects of the experimental stations that will be particularly attractive for chemistry research. SwissFEL is a revolutionary new machine that will complement and extend the time-resolved chemistry efforts in the Swiss research community.

Mixtures of different particle species are often investigated using the angular averages of the scattered X-ray intensity. The number of species is deduced by singular value decomposition methods. The full disentanglement of the data into per-species contributions requires additional knowledge about the system under investigation. We propose to exploit higher-order angular X-ray intensity correlations with a new computational protocol, which we apply to synchrotron data from two-species mixtures of two-dimensional static test nanoparticles. Without any other information besides the correlations, we demonstrate the assessment of particle species concentrations in the measured data sets, as well as the full ab initio reconstruction of both particle structures. The concept extends straightforwardly to more species and to the three-dimensional case, whereby the practical application will require the measurements to be performed at an X-ray free electron laser.

We report on the fabrication of identical gold nanoparticles for X-ray scattering test experiments with synchrotron radiation. For this purpose, direct-write e-beam lithography followed by gold electroplating techniques were applied, which produced large numbers of nanoparticles with the chosen complex shape and overall size between 200 and 500nm. We prepared two sorts of samples: nanostructures grown in random position and orientation on a membrane mimicking a 2D ''liquid'' and nanostructures detached from their substrate and suspended in glycerol. The key for the success of the X-ray experiments was the extremely high shape and size homogeneity of the particles generated top-down. We also demonstrated that exposing molds into multi-layered e-beam resists opens further possibilities for the shape control of the electroplated nanoparticles along their growth direction. We discuss technological aspects of the fabrication procedure in detail and show examples of various fabricated nanoparticles.

Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented., X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

Optica 5(12), 1521 - 1524 (2018). doi:10.1364/OPTICA.5.001521, High-brilliance x-ray sources (x-ray free-electron lasers or diffraction-limited storage rings) allow the visualization of ultrafast processes in a 2D manner using single exposures. Current 3D approaches scan the sample using multiple exposures, and hence they are not compatible with single-shot acquisitions. Here we propose and verify experimentally an x-ray multi-projection imaging approach, which uses a crystal to simultaneously acquire nine angularly resolved projections with a single x-ray exposure. When implemented at high-brilliance sources, this approach can provide volumetric information of natural processes and non-reproducible samples in the micrometer to nanometer resolution range, and resolve timescales from microseconds down to femtoseconds., Published by OSA, Washington, DC

NMR-Profiles are quantitative one-dimensional (1D) presentations of 2D [¹⁵N, ¹H]-correlation spectra used to monitor the quality of protein solutions prior to and during NMR structure determinations and functional studies. In our current use in structural genomics projects, an NMR-Profile is recorded at the outset of a structure determination, using a uniformly ¹⁵N-labeled microscale sample of the protein. We thus assess the extent to which polypeptide backbone resonance assignments can be achieved with given NMR techniques, for example, conventional triple resonance experiments or APSY-NMR. With the availability of sequence-specific polypeptide backbone resonance assignments in the course of the structure determination, an "Assigned NMR-Profile" is generated, which visualizes the variation of the ¹⁵N - ¹H correlation cross peak intensities along the sequence and thus maps the sequence locations of polypeptide segments for which the NMR line shapes are affected by conformational exchange or other processes. The Assigned NMR-Profile provides a guiding reference during later stages of the structure determination, and is of special interest for monitoring the protein during functional studies, where dynamic features may be modulated during physiological processes.

Euplotes is diversified into dozens of widely distributed species that produce structurally homologous families of water-borne protein pheromones governing self-/nonself-recognition phenomena. Structures of pheromones and pheromone coding genes have so far been studied from species lying in different positions of the Euplotes phylogenetic tree. We have now cloned the coding genes and determined the NMR molecular structure of four pheromones isolated from Euplotes petzi, a polar species which is phylogenetically distant from previously studied species and forms the deepest branching clade in the tree. The E. petzi pheromone genes have significantly shorter sequences than in other congeners, lack introns, and encode products of only 32 amino acids. Likewise, the three-dimensional structure of the E. petzi pheromones is markedly simpler than the three-helix up-down-up architecture previously determined in another polar species, Euplotes nobilii, and in a temperate-water species, Euplotes raikovi. Although sharing the same up-down-up architecture, it includes only two short α-helices that find their topological counterparts with the second and third helices of the E. raikovi and E. nobilii pheromones. The overall picture that emerges is that the evolution of Euplotes pheromones involves progressive increases in the gene sequence length and in the complexity of the three-dimensional molecular structure.

Ciliates communicate via diffusible signaling pheromones that have been identified in association with genetic mechanisms of mating types, either binary or multiple, that control the ciliate-specific sexual phenomenon of conjugation. Except for a glycoprotein and a tryptophan-related molecule acting as pheromones in Blepharisma, the other pheromones are cysteine-rich proteins varying in length among Euplotes species from 32 to 109 amino acid residues. These proteins are all structurally homologous sharing a common three-helix bundle fold, which allows them to bind cells in competition with one another in close analogy with the families of protein growth factors and cytokines of multicellular organisms. Although identified in the extracellular environment for their mating-induction activity, pheromones act also, and presumably primarily, as autocrine growth factors that bind to, and promote the reproduction of the same cells from which they are constitutively released.

Next-generation X-ray sources, based on the X-ray Free Electron Laser (XFEL) concept, will provide highly coherent, ultrashort pulses of soft and hard X-rays with peak intensity many orders of magnitude above that of a synchrotron. These pulses will allow studies of femtosecond dynamics at nanometer resolution and with chemical selectivity. They will produce coherent-diffraction images of organic and inorganic nanostructures without the deleterious effects of radiation damage.

The NMR structures of the TM1112 and TM1367 proteins from Thermotoga maritima in solution at 298 K were determined following a new protocol which uses the software package UNIO for extensive automation. The results obtained with this novel procedure were evaluated by comparison with the crystal structures solved by the JCSG at 100 K to 1.83 and 1.90 Å resolution, respectively. In addition, the TM1112 solution structure was compared with an NMR structure solved by the NESG using a conventional largely interactive methodology. For both proteins, the newly determined NMR structure could be superimposed with the crystal structure with r.m.s.d. values of, Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 66 (10), ISSN:1744-3091, ISSN:2053-230X

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for “middle of the SARS-unique domain”) in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.

Sequence-specific NMR assignments of the globular core comprising the residues 1066-1181 within the non-structural protein nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [(13)C, (15)N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure determination. A preliminary identification of regular secondary structures has been derived from the (13)C chemical shifts.

Sequence-specific NMR assignments of an internal domain of the protein nsp3, nsp3(513-651), which is a part of the SARS coronavirus (SARS-CoV) replicase polyprotein, have been determined, using triple-resonance NMR experiments with the uniformly [(13)C,(15)N]-labeled protein. The complete assignments (>99%) provide the basis for the ongoing three-dimensional structure determination.

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear (1)H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-alpha-helix bundle with an up-down-up topology and a 3(10)-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters.

Structural dynamics 2(4), 041718 (2015). doi:10.1063/1.4922774, Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination., Published by AIP Publishing LLC, Melville, NY

The NMR structure of the 206-residue protein NP_346487.1 was determined with the J-UNIO protocol, which includes extensive automation of the structure determination. With input from three APSY-NMR experiments, UNIO-MATCH automatically yielded 77 % of the backbone assignments, which were interactively validated and extended to 97 %. With an input of the near-complete backbone assignments and three 3D heteronuclear-resolved [1H,1H]-NOESY spectra, automated side chain assignment with UNIO-ATNOS/ASCAN resulted in 77 % of the expected assignments, which was extended interactively to about 90 %. Automated NOE assignment and structure calculation with UNIO-ATNOS/CANDID in combination with CYANA was used for the structure determination of this two-domain protein. The individual domains in the NMR structure coincide closely with the crystal structure, and the NMR studies further imply that the two domains undergo restricted hinge motions relative to each other in solution. NP_346487.1 is so far the largest polypeptide chain to which the J-UNIO structure determination protocol has successfully been applied., Journal of Biomolecular NMR, 61 (1), ISSN:0925-2738, ISSN:1573-5001

A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [1H,1H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination., Journal of Biomolecular NMR, 61 (1), ISSN:0925-2738, ISSN:1573-5001