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Scientific data 3, 160064 -(2016). doi:10.1038/sdata.2016.64, Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here., Published by Nature Publ. Group, London

We report on the use of multilayer Laue lenses to focus the intense X-ray Free Electron Laser (XFEL) beam at the European XFEL to a spot size of a few tens of nanometers. We present the procedure to align and characterize these lenses and discuss challenges working with the pulse trains from this unique x-ray source.

Communications Physics 3(1), 97 (2020). doi:10.1038/s42005-020-0362-y, The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate., Published by Springer Nature, London

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.

64th Annual Biophysical Society Meeting, San Diego, 15 Feb 2020 - 19 Feb 2020; Biophysical journal 118(3), 137a - 138a (2020). doi:10.1016/j.bpj.2019.11.877, Outrunning radiation damage, highly intense femtosecond pulses of X-ray free-electron lasers (XFELs) open up the possibility of structure determination of macromolecules to viruses at room temperature, by aggregating diffraction patterns recorded from uncrystallized single-particles. A key challenge in XFEL single-particle diffractive imaging (SPI) is to either constrain the orientation of the particle or to determine it from each of the very noisy weak diffraction patterns. Here we report a unique approach to address these challenges using structural DNA nanotechnology. A DNA-origami ���rigid tail��� is site-specifically attached to the macromolecule to flow-align it in a thin liquid jet, and also provides a strong holographic reference. In a proof-of-principle study, the computational design and production of the DNA-origami-target construct has been achieved and the experimental results obtained from the Linac Coherent Light Source (LCLS), USA show the alignment of the target single-particle, consistent with simulations, at extremely low concentrations approaching single-molecule in the interaction region with the nanofocus hard X-ray laser beam, in a sum of as few as a thousand single-shots. The results open up the possibility of single-molecule diffractive imaging in solution with XFEL pulses. Acknowledgements: The Human Frontier Science Program (RGP0010/2017)., Published by Soc., Bethesda, Md.

Optics express 27(26), 37816 - (2019). doi:10.1364/OE.27.037816, An outstanding question in X-ray single particle imaging experiments has been thefeasibility of imaging sub 10-nm-sized biomolecules under realistic experimental conditionswhere very few photons are expected to be measured in a single snapshot and instrumentbackground may be significant relative to particle scattering. While analyses of simulated datahave shown that the determination of an average image should be feasible using Bayesian methodssuch as the EMC algorithm, this has yet to be demonstrated using experimental data containingrealistic non-isotropic instrument background, sample variability and other experimental factors.In this work, we show that the orientation and phase retrieval steps work at photon counts dilutedto the signal levels one expects from smaller molecules or with weaker pulses, using data fromexperimental measurements of 60-nm PR772 viruses. Even when the signal is reduced to afraction as little as 1/256, the virus electron density determined using ab initio phasing is ofalmost the same quality as the high-signal data. However, we are still limited by the total numberof patterns collected, which may soon be mitigated by the advent of high repetition-rate sourceslike the European XFEL and LCLS-II., Published by Soc., Washington, DC

The application of a new generation of x-ray sources called X-ray Free Electron Lasers (XFELs) to diffractive imaging has allowed structural studies of specimens not previously accessible. Specimens of reduced crystallinity are of particular interest, including fibrous nano-crystals and single fibrous molecules. Diffractive imaging experiments using XFELs generate large datasets of diffraction frames from specimens with random, unknown orientations. The orientation of each diffraction frame needs to be determined from features in the pattern in order to register and merge the dataset for subsequent structural analysis. Certain sample delivery techniques simplify this process by limiting the range of orientations a specimen may take. In this paper we consider two sample delivery techniques: a liquid jet and a fixed target on a silicon wafer. Orientations determined from diffraction patterns from each delivery method are classified in order to investigate the type of orientation present. This information also helps to characterize the quality of sample preparations and provides feedback valuable for designing future experiments.

The application of high-powered X-ray Free Electron Lasers (XFELs) in Serial Femtosecond Crystallography (SFX) has led to an increasing number of crystal structure determinations. Achieving the same success for systems of diminished crystallinity, such as fibrous systems and single particles, is inhibited by low signal strength and difficulties in orientating the data. We present methods developed for analyzing serial diffraction data from fibrous systems. The data are processed to identify cases where a single XFEL pulse intersects a single fibril. The fibril orientations are then determined by analysis of detected features within the diffraction data. With the fibril orientation determined, serial diffraction data is merged in 3D reciprocal space, allowing the individual structure amplitudes to be calculated. This allows structural studies of previously inaccessible fibrous systems to be performed, and represents a step closer towards the long-term goal of imaging single particles.

Physical review letters 119(5), 053401 (2017). doi:10.1103/PhysRevLett.119.053401, Established x-ray diffraction methods allow for high-resolution structure determination of crystals, crystallized protein structures, or even single molecules. While these techniques rely on coherent scattering, incoherent processes like fluorescence emission—often the predominant scattering mechanism—are generally considered detrimental for imaging applications. Here, we show that intensity correlations of incoherently scattered x-ray radiation can be used to image the full 3D arrangement of the scattering atoms with significantly higher resolution compared to conventional coherent diffraction imaging and crystallography, including additional three-dimensional information in Fourier space for a single sample orientation. We present a number of properties of incoherent diffractive imaging that are conceptually superior to those of coherent methods., Published by APS, College Park, Md.

Scientific data 4, 170079 (2017). doi:10.1038/sdata.2017.79, Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single- particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency., Published by Macmillan, London

The application of newly developed imaging techniques using X-ray Free Electron Laser (XFEL) sources requires equally new methods for analysing the large volumes of data collected. Methods are described for processing and assessing the quality of the measured XFEL diffraction patterns. The methods are applied to an XFEL data-set to extract diffraction patterns that show strong molecular alignment.

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.]

The Single Particles, Clusters and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL is located behind the SASE1 undulator and aims to support imaging and structure determination of biological specimen between about 0.1 μm and 1 μm size. The instrument is designed to work at photon energies from 3 keV up to 16 keV. Here, we propose a cost-effective proof-of-principle experiment, aiming to demonstrate the actual feasibility of a single molecule diffraction experiment at the European XFEL. To this end, we assume self-seeding capabilities at SASE1 and we suggest to make use of the baseline European XFEL accelerator complex—with the addition of a slotted-foil setup—and of the SPB/SFX instrument. As a first step towards the realization of an actual experiment, we developed a complete package of computational tools for start-to-end simulations predicting its performance. Single biomolecule imaging capabilities at the European XFEL can be reached by exploiting special modes of operation of the accelerator complex and of the SASE1 undulator. The output peak power can be increased up to more than 1.5 TW, which allows to relax the requirements on the focusing efficiency of the optics and to reach the required fluence without changing the present design of the SPB/SFX instrument. Explicit simulations are presented using the 15-nm size RNA Polymerase II molecule as a case study. Noisy diffraction patterns were generated and they were processed to generate the 3D intensity distribution. We discuss requirements to the signal-to-background ratio needed to obtain a correct pattern orientation. When these are fulfilled, our results indicate that one can achieve diffraction without destruction with about 0.1 photons per Shannon pixel per shot at 4 Å resolution with 10$^{13}$ photons in a 4 fs pulse at 4 keV photon energy and in a 0.3 μm focus, corresponding to a fluence of 10$^{14}$ photons/μm$^{2}$. We assume negligible structured background. At this signal level, one needs only about 30 000 diffraction patterns to recover full 3D information. At the highest repetition rate manageable by detectors at European XFEL, one will be able to accumulate these data within a fraction of an hour, even assuming a relatively low hit probability of about a percent.

Single-particle imaging experiments of biomolecules at x-ray free-electron lasers (XFELs) require processing hundreds of thousands of images that contain very few x-rays. Each low-fluence image of the diffraction pattern is produced by a single, randomly oriented particle, such as a protein. We demonstrate the feasibility of recovering structural information at these extremes using low-fluence images of a randomly oriented 2D x-ray mask. Successful reconstruction is obtained with images averaging only 2.5 photons per frame, where it seems doubtful there could be information about the state of rotation, let alone the image contrast. This is accomplished with an expectation maximization algorithm that processes the low-fluence data in aggregate, and without any prior knowledge of the object or its orientation. The versatility of the method promises, more generally, to redefine what measurement scenarios can provide useful signal.

Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7 keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 μm x 1.7 μm full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experimental parameters, and provide a basis for developing algorithms for image analysis and reconstruction., Measurement(s) X-ray diffraction data Technology Type(s) free electron laser Sample Characteristic - Organism Enterobacteria phage PR772 Sample Characteristic - Location contiguous United States of America Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13070546