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Methods are described for processing XFEL data from individual crystalline fibrils. The methods are applied to data collected at the Linac Coherent Light Source from an amyloid-forming oligopeptide from the adenovirus shaft., Serial diffraction data collected at the Linac Coherent Light Source from crystalline amyloid fibrils delivered in a liquid jet show that the fibrils are well oriented in the jet. At low fibril concentrations, diffraction patterns are recorded from single fibrils; these patterns are weak and contain only a few reflections. Methods are developed for determining the orientation of patterns in reciprocal space and merging them in three dimensions. This allows the individual structure amplitudes to be calculated, thus overcoming the limitations of orientation and cylindrical averaging in conventional fibre diffraction analysis. The advantages of this technique should allow structural studies of fibrous systems in biology that are inaccessible using existing techniques.

Ductal carcinoma in situ (DCIS) is a heterogeneous disease, for which the best adjuvant treatment is still uncertain. Many attempts of risk-groups stratification have been made over time, developing prognostic scores to predict risk of local recurrence (LR) on the basis of features such as age, final surgical margins (FSM) status, grade, and tumor size. The aim of our analysis was to evaluate the patterns of recurrence from a two large-institutional retrospective series.We collected data on 457 patients treated with BCS and adjuvant RT between 1990 and 2012. Final analysis was performed on 278 patients, due to missing data about hormonal status (HS). Patients were treated at the Radiation Oncology Unit of the University of Florence (n = 195), and S. Maria Annunziata Hospital (n = 83) (Florence, Italy).At a median follow up time of 10.8 years (range 3-25), we observed 20 LR (7.2%). The 5-year and 10-year LR rates were 4.9% and 10.2%, respectively. At Cox regression univariate analysis, estrogen receptor (ER) positive status (p = 0.001), HS positive (p = 0.003), and FSM1 mm (p = 0.0001) significantly impacted on LR. At Cox regression multivariate analysis positive ER status maintained a protective role (p = 0.003), and FSM status1 mm its negative impact (p = 0.0001) on LR rate.Our experience confirmed the wide heterogeneity of DCIS. Inadequate FSM and negative ER status negatively influenced LR rates. Tumor biology should be integrated in adjuvant treatment decision-making process.

Summary 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.

Serial femtosecond crystallography (SFX) is an emerging method for data collection at free-electron lasers (FELs) in which single diffraction snapshots are taken from a large number of crystals. The partial intensities collected in this way are then combined in a scheme called Monte Carlo integration, which provides the full diffraction intensities. However, apart from having to perform this merging, the Monte Carlo integration must also average out all variations in crystal quality, crystal size, X-ray beam properties and other factors, necessitating data collection from thousands of crystals. Because the pulses provided by FELs running in the typical self-amplified spontaneous emission (SASE) mode of operation have very irregular, spiky spectra that vary strongly from pulse to pulse, it has been suggested that this is an important source of variation contributing to inaccuracies in the intensities, and that, by using monochromatic pulses produced through a process called self-seeding, fewer images might be needed for Monte Carlo integration to converge, resulting in more accurate data. This paper reports the results of two experiments performed at the Linac Coherent Light Source in which data collected in both SASE and self-seeded mode were compared. Importantly, no improvement attributable to the use of self-seeding was detected. In addition, other possible sources of variation that affect SFX data quality were investigated, such as crystal-to-crystal variations reflected in the unit-cell parameters; however, these factors were found to have no influence on data quality either. Possibly, there is another source of variation as yet undetected that affects SFX data quality much more than any of the factors investigated here.

Proceedings of the National Academy of Sciences of the United States of America 114(9), 2247 – 2252 (2017). doi:10.1073/pnas.1609243114, To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm$^3$ in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm$^3$ in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach., Published by National Acad. of Sciences, Washington, DC

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere1. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies3, 4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

The room-temperature structure of lysozyme is determined using 40000 individual diffraction patterns from micro-crystals flowing in liquid suspension across a synchrotron microfocus beamline., A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Å resolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.

Anomalous signal from sulfur atoms present in most proteins was used for de novo phasing of XFEL data and solving a GPCR structure., Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A2A adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.

Nature 530(7589), 202 - 206(2016). doi:10.1038/nature16949, The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 $\mathring{A}$ limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 $\mathring{A}$ as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 $\mathring{A}$. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing., Published by Nature Publ. Group, London [u.a.]

Developments in the CrystFEL software suite, for processing diffraction data from ‘serial crystallography’ experiments, are described., CrystFEL is a suite of programs for processing data from ‘serial crystallography’ experiments, which are usually performed using X-ray free-electron lasers (FELs) but also increasingly with other X-ray sources. The CrystFEL software suite has been under development since 2009, just before the first hard FEL experiments were performed, and has been significantly updated and improved since then. This article describes the most important improvements which have been made to CrystFEL since the first release version. These changes include the addition of new programs to the suite, the ability to resolve ‘indexing ambiguities’ and several ways to improve the quality of the integrated data by more accurately modelling the underlying diffraction physics.

We report on the analysis of virtual powder-diffraction patterns from serial femtosecond crystallography (SFX) data collected at an X-ray free-electron laser. Different approaches to binning and normalizing these patterns are discussed with respect to the microstructural characteristics which each highlights. Analysis of SFX data from a powder of Pr0.5Ca0.5MnO3 in this way finds evidence of other trace phases in its microstructure which was not detectable in a standard powder-diffraction measurement. Furthermore, a comparison between two virtual powder pattern integration strategies is shown to yield different diffraction peak broadening, indicating sensitivity to different types of microstrain. This paper is a first step in developing new data analysis methods for microstructure characterization from serial crystallography data.

The photon density profile of an X-ray free-electron laser (XFEL) beam at the focal position is a critical parameter for serial femtosecond crystallography (SFX), but is difficult to measure because of the destructive power of the beam. A novel high intensity radiation induced phasing method (HIRIP) has been proposed as a general experimental approach for protein structure determination, but has proved to be sensitive to variations of the X-ray intensity, with uniform incident fluence desired for best performance. Here we show that experimental SFX data collected at the nano-focus chamber of the Coherent X-ray Imaging end-station at the Linac Coherent Light Source using crystals with a limited size distribution suggests an average profile of the X-ray beam that has a large variation of intensity. We propose a new method to improve the quality of high fluence data for HI-RIP, by identifying and removing diffraction patterns from crystals exposed to the low intensity region of the beam. The method requires crystals of average size comparable to the width of the focal spot.

Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.

Analysis of serial femtosecond crystallography data collected at the Linac Coherent Light Source using two distinct photon fluxes shows different degrees of ionization of Gd atoms bound to a lysozyme protein, due to electronic damage processes. The charge contrast on the heavy atoms is quantified using difference Fourier maps, and the way in which this could be applied to phasing is discussed., X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential ‘bleaching’ of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

We use a Mach-Zehnder type autocorrelator to split and delay XUV pulses from the FLASH soft X-ray laser for triggering and subsequently probing the explosion of aerosolised sugar balls. FLASH was running at 182 eV photon energy with pulses of 70 fs duration. The delay between the pump-probe pulses was varied between zero and 5 ps, and the pulses were focused to reach peak intensities above 1016 W/cm2 with an off-axis parabola. The direct pulse triggered the explosion of single aerosolised sucrose nano-particles, while the delayed pulse probed the exploding structure. The ejected ions were measured by ion time of flight spectrometry, and the particle sizes were measured by coherent diffractive imaging. The results show that sucrose particles of 560-1000 nm diameter retain their size for about 500 fs following the first exposure. Significant sample expansion happens between 500 fs and 1 ps. We present simulations to support these observations.

Here, it is shown that simulated native serial femtosecond crystallography (SFX) cathepsin B data can be phased by rapid ionization of sulfur atoms. Utilizing standard software adopted for radiation-damage-induced phasing (RIP), the effects on both substructure determination and phasing of the number of collected patterns and fluences are explored for experimental conditions already available at current free-electron laser facilities.

We investigate the electronic structure of the Sr${}_{n+1}$Ru${}_{n}$O${}_{3n+1}$ family $(n=1,2,3)$ in the vicinity the Fermi level by means of polarization-dependent resonant O 1$s$ x-ray emission spectroscopy. Using both energy window and polarization analysis we disentangle the contribution of apical and planar oxygen, and compare it with first-principles calculations. Our results provide a new insight on the nature of the oxygen bonds and the role of Sr $3d$ states.

Diffraction Before Destruction A bottleneck in x-ray crystallography is the growth of well-ordered crystals large enough to obtain high-resolution diffraction data within an exposure that limits radiation damage. Serial femtosecond crystallography promises to overcome these constraints by using short intense pulses that out-run radiation damage. A stream of crystals is flowed across the free-electron beam and for each pulse, diffraction data is recorded from a single crystal before it is destroyed. Redecke et al. (p. 227 , published online 29 November; see the Perspective by Helliwell ) used this technique to determine the structure of an enzyme from Trypanosoma brucei , the parasite that causes sleeping sickness, from micron-sized crystals grown within insect cells. The structure shows how this enzyme, which is involved in degradation of host proteins, is natively inhibited prior to activation, which could help in the development of parasite-specific inhibitors.

Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure., Serial femtosecond crystallography is an X-ray free-electron-laser-based method that uses X-ray bursts to determine protein structures. Here the authors present the structure of a photosynthetic reaction centre, an integral membrane protein, achieved with no sign of X-ray-induced radiation damage.

The layer-dependent Debye temperature of Ru(0001) is determined by means of high-energy resolution core-level photoelectron spectroscopy measurements. The possibility to disentangle three different components in the Ru 3d{sub 5/2} spectrum of Ru(0001), originating from bulk, first-, and second-layer atoms, allowed us to follow the temperature evolution of their photoemission line shapes and binding energies. Temperature effects were detected, namely, a lattice thermal expansion and a layer-dependent phonon broadening, which was interpreted within the framework of the Hedin-Rosengren formalism based on the Debye theory. The resulting Debye temperature of the top-layer atoms is 295{+-}10 K, lower than that of the bulk (T=668{+-}5 K) and second-layer (T=445{+-}10 K) atoms. While these results are in agreement with the expected phonon softening at the surface, we show that a purely harmonic description of the motion of the surface atoms is not valid, since anharmonic effects contribute significantly to the position and line shape of the different core-level components.