Fluctuation X-ray scattering (FXS) is an emerging experimental technique in which solution scattering data are collected using X-ray exposures below rotational diffusion times, resulting in angularly anisotropic X-ray snapshots that provide several orders of magnitude more information than traditional solution scattering data. Such experiments can be performed using the ultrashort X-ray pulses provided by a free-electron laser source, allowing one to collect a large number of diffraction patterns in a relatively short time. Here, we describe a test data set for FXS, obtained at the Linac Coherent Light Source, consisting of close to 100 000 multi-particle diffraction patterns originating from approximately 50 to 200 Paramecium Bursaria Chlorella virus particles per snapshot. In addition to the raw data, a selection of high-quality pre-processed diffraction patterns and a reference SAXS profile are provided.

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.

Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe-4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.

Femtosecond X-ray pulses were used to obtain diffraction data on photosystem II, revealing conformational changes as the complex transitions from the dark S1 state to the double-pumped S3 state; the time-resolved serial femtosecond crystallography technique enables structural determination of protein conformations that are highly prone to traditional radiation damage. It has recently been shown that extremely short and intense radiation pulses from X-ray free-electron lasers can be used to obtain diffraction data on nanometre- to micrometre-sized protein crystals before the crystal suffers radiation damage. The hope is that this 'serial femtosecond crystallography' (SFX) approach will produce structures of proteins and protein complexes that do not yield well-ordered macroscopic crystals. These authors collected time-resolved SFX data on small crystals of photosystem II of photosynthesis during its transition from the 'dark' S1 state to the double-excited S3 state. At present the resolution of this technique is moderate, but it is sufficient to reveal significant conformational changes at the Mn4CaO5 cluster at the heart of the oxygen evolving complex and at the electron acceptor site. 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.

Single shot diffraction imaging experiments via X-ray free-electron lasers can generate as many as hundreds of thousands of diffraction patterns of scattering objects. Recovering the real space contrast of a scattering object from these patterns currently requires a reconstruction process with user guidance in a number of steps, introducing severe bottlenecks in data processing. We present a series of measures that replace user guidance with algorithms that reconstruct contrasts in an unsupervised fashion. We demonstrate the feasibility of automating the reconstruction process by generating hundreds of contrasts obtained from soot particle diffraction experiments.

Characterizing intense, focused x-ray free electron laser (FEL) pulses is crucial for their use in diffractive imaging.We describe how the distribution of average phase tilts and intensities on hard x-ray pulses with peak intensities of 10 21 W/m 2 can be retrieved from an ensemble of diffraction patterns produced by 70 nm-radius polystyrene spheres, in a manner that mimics wavefront sensors.Besides showing that an adaptive geometric correction may be necessary for diffraction data from randomly injected sample sources, our paper demonstrates the possibility of collecting statistics on structured pulses using only the diffraction patterns they generate and highlights the imperative to study its impact on single-particle diffractive imaging.

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.

Coherent diffractive imaging with x-ray free-electron lasers (XFEL) promises high-resolution structure determination of noncrystalline objects. Randomly oriented particles are exposed to XFEL pulses for acquisition of two-dimensional (2D) diffraction snapshots. The knowledge of their orientations enables 3D imaging by multiview reconstruction, combining 2D diffraction snapshots in different orientations. Here we introduce a globally optimal algorithm that can infer these orientations. We apply it to experimental XFEL data of nanoparticles and so determine their 3D electron density.

X-ray free-electron lasers (FELs) enable crystallographic data collection using extremely bright femtosecond pulses from microscopic crystals beyond the limitations of conventional radiation damage. This diffraction-before-destruction approach requires a new crystal for each FEL shot and, since the crystals cannot be rotated during the X-ray pulse, data collection requires averaging over many different crystals and a Monte Carlo integration of the diffraction intensities, making the accurate determination of structure factors challenging. To investigate whether sufficient accuracy can be attained for the measurement of anomalous signal, a large data set was collected from lysozyme microcrystals at the newly established `multi-purpose spectroscopy/imaging instrument' of the SPring-8 Ångstrom Compact Free-Electron Laser (SACLA) at RIKEN Harima. Anomalous difference density maps calculated from these data demonstrate that serial femtosecond crystallography using a free-electron laser is sufficiently accurate to measure even the very weak anomalous signal of naturally occurring S atoms in a protein at a photon energy of 7.3 keV.

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

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We describe femtosecond X-ray diffraction data sets of viruses and nanoparticles collected at the Linac Coherent Light Source.The data establish the first large benchmark data sets for coherent diffraction methods freely available to the public, to bolster the development of algorithms that are essential for developing this novel approach as a useful imaging technique.Applications are 2D reconstructions, orientation classification and finally 3D imaging by assembling 2D patterns into a 3D diffraction volume.

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

Profiling structured beams produced by X-ray free-electron lasers (FELs) is crucial to both maximizing signal intensity for weakly scattering targets and interpreting their scattering patterns. Earlier ablative imprint studies describe how to infer the X-ray beam profile from the damage that an attenuated beam inflicts on a substrate. However, the beams in-situ profile is not directly accessible with imprint studies because the damage profile could be different from the actual beam profile. On the other hand, although a Shack-Hartmann sensor is capable of in-situ profiling, its lenses may be quickly damaged at the intense focus of hard X-ray FEL beams. We describe a new approach that probes the in-situ morphology of the intense FEL focus. By studying the translations in diffraction patterns from an ensemble of randomly injected sub- micron latex spheres, we were able to determine the non-Gaussian nature of the intense FEL beam at the Linac Coherent Light Source (SLAC National Laboratory) near the FEL focus. We discuss an experimental application of such a beam-profiling technique, and the limitations we need to overcome before it can be widely applied.

The Max Planck Advanced Si:udy Group (ASG) at the Center for Free Electron Laser Science (CFEL) has created the CFEL-ASG Software Suite CASS to view, process and analyse multi-parameter experimental data acquired at Free Electron Lasers (FELs) using the CFEL-ASG Multi Purpose (CAMP) instrument Struder et al. (2010) [6].The software is based on a modular design so that it can be adjusted to accommodate the needs of all the various experiments that are conducted with the CAMP instrument. In fact, this allows the use of the software in all experiments where multiple detectors are involved. One of the key aspects of CASS is that it can be used either 'on-line', using a live data stream from the free-electron laser facility's data acquisition system to guide the experiment, and 'off-line', on data acquired from a previous experiment which has been saved to file.Program summaryProgram title: CASSCatalogue identifier: AEMP_v1_0Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AEMP_v1_0.htmlProgram obtainable from: CPC Program Library, Queen's University, Belfast, N. IrelandLicensing provisions: GNU General Public Licence, version 3No. of lines in distributed program, including test data, etc.: 167073No. of bytes in distributed program, including test data, etc.: 1065056Distribution format: tar.gzProgramming language: C + +.Computer: Intel x86-64.Operating system: GNU/Linux (for information about restrictions see outlook).RAM: >8 GBClassification: 2.3, 3, 15, 16.4.External routines: Qt-Framework[1], SOAP[2], (optional HDF5[3], VIGRA[4], ROOT[5], QWT[6])Nature of problem: Analysis and visualisation of scientific data acquired at Free-Electron-LasersSolution method: Generalise data access and storage so that a variety of small programming pieces can be linked to form a complex analysis chain.Unusual features: Complex analysis chains can be built without recompiling the programAdditional comments: An updated extensive documentation of CASS is available at [7].Running time: Depending on the data size and complexity of analysis algorithms.References:[1] http://qt.nokia.com[2] http://www.cs.fsu.edu/-engelen/soap.html[3] http://www.hdfgroup.org/HDF5/[4] http://hci.iwr.uni-heidelberg.de/vigra/[5] http://root.cern.ch[6] http://qwt.sourceforge.net/[7] http://www.mpi-hd.mpg.de/personalhomes/gitasg/cass (C) 2012 Elsevier B.V. All rights reserved.