We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates from the expected perfect icosahedral symmetry. Our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.

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.

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.

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.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.