Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals.

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.

Numerical simulations of the hard X-ray pulse intensity distribution at the Linac Coherent Light Source.

Numerical simulations of the current and future pulse intensity distributions at selected locations along the Far Experimental Hall, the hard X-ray section of the Linac Coherent Light Source (LCLS), are provided. Estimates are given for the pulse fluence, energy and size in and out of focus, taking into account effects due to the experimentally measured divergence of the X-ray beam, and measured figure errors of all X-ray optics in the beam path. Out-of-focus results are validated by comparison with experimental data. Previous work is expanded on, providing quantitatively correct predictions of the pulse intensity distribution. Numerical estimates in focus are particularly important given that the latter cannot be measured with direct imaging techniques due to detector damage. Finally, novel numerical estimates of improvements to the pulse intensity distribution expected as part of the on-going upgrade of the LCLS X-ray transport system are provided. We suggest how the new generation of X-ray optics to be installed would outperform the old one, satisfying the tight requirements imposed by X-ray free-electron laser facilities.

Microfield dynamics in dense hydrogen plasmas with high-Z impurities.

We use large-scale classical molecular dynamics to determine microfield properties for several dense plasma mixtures. By employing quantum statistical potentials (QSPs) to regularize the Coulomb interaction, our simulations follow motions of electrons as well as ions for times long enough to track relaxation phenomena involving both types of particles. Coulomb coupling, relative to temperature, of different pairs of species in the hot, dense matter being simulated ranges from weak to strong. We first study the effect of such coupling differences, along with composition and QSP differences, on the roles of electrons and various mixture components in determining probability distributions of instantaneous, total microfields experienced by the ions. Then, we address two important dynamical questions: (1) How is the quasistatic part of the total field to be extracted from the time-dependent simulation data? (2) Under what conditions does the commonly used approximation of ions with fixed Yukawa-like screening by free electrons accurately describe quasistatic fields? We identify a running, short-time average of the total field at each ion as its slowly evolving, quasistatic part. We consider several ways to specify the averaging interval, and note the influence of ion dynamics in this issue. When all species are weakly coupled, the quasistatic fields have probability distributions agreeing well with those we obtain from simulations of Yukawa-screened ions. However, agreement deteriorates as the coupling between high-Z ions increases well beyond unity, principally because the Yukawa model tends to underestimate the true screening of close high-Z pairs. Examples of this fact are given, and some consequences for the high-field portions of probability distributions are discussed.

Aperiodic Mo/Si multilayers for hard x-rays.

In this work we have developed aperiodic Molybdenum/Silicon (Mo/Si) multilayers (MLs) to reflect 16.25 keV photons at a grazing angle of incidence of 0.6° ± 0.05°. To the best of our knowledge this is the first time this material system has been used to fabricate aperiodic MLs for hard x-rays. At these energies new hurdles arise. First of all a large number of bilayers is required to reach saturation. This poses a challenge from the manufacturing point of view, as thickness control of each ML period becomes paramount. The latter is not well defined a priori, due to the thickness of the interfacial silicide layers which has been observed to vary as a function of Mo and Si thickness. Additionally an amorphous-to-crystalline transition for Mo must be avoided in order maintain reasonably low roughness at the interfaces. This transition is well within the range of thicknesses pertinent to this study. Despite these difficulties our data demonstrates that we achieved reasonably flat ML response across the angular acceptance of ± 0.05°, with an experimentally confirmed average reflectivity of 28%. Such a ML prescription is well suited for applications in the field of hard x-ray imaging of highly diverging sources.

Effect of slope errors on the performance of mirrors for x-ray free electron laser applications.

In this work we point out that slope errors play only a minor role in the performance of a certain class of x-ray optics for X-ray Free Electron Laser (XFEL) applications. Using physical optics propagation simulations and the formalism of Church and Takacs [Opt. Eng. 34, 353 (1995)], we show that diffraction limited optics commonly found at XFEL facilities posses a critical spatial wavelength that makes them less sensitive to slope errors, and more sensitive to height error. Given the number of XFELs currently operating or under construction across the world, we hope that this simple observation will help to correctly define specifications for x-ray optics to be deployed at XFELs, possibly reducing the budget and the timeframe needed to complete the optical manufacturing and metrology.

Reproducible radiation-damage processes in proteins irradiated by intense x-ray pulses.

X-ray free-electron lasers have enabled femtosecond protein nanocrystallography, a novel method to determine the structure of proteins. It allows time-resolved imaging of nanocrystals that are too small for conventional crystallography. The short pulse duration helps in overcoming the detrimental effects of radiation damage because x rays are scattered before the sample has been significantly altered. It has been suggested that, fortuitously, the diffraction process self-terminates abruptly once radiation damage destroys the crystalline order. Our calculations show that high-intensity x-ray pulses indeed trigger a cascade of damage processes in ferredoxin crystals, a particular metalloprotein of interest. However, we found that the damage process is initially not completely random. Correlations exist among the protein monomers, so that Bragg diffraction still occurs in the damaged crystals, despite significant atomic displacements. Our results show that the damage process is reproducible to a certain degree, which is potentially beneficial for the orientation step in single-molecule imaging.

Fixed-target protein serial microcrystallography with an x-ray free electron laser.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser.

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 atmosphere. 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 developed 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 studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Femtosecond X-ray diffraction from two-dimensional protein crystals.

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 Šresolution 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.

7 Å resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source.

Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump-probe experiments at subpicosecond time resolution.

Nonequilibrium electron dynamics in materials driven by high-intensity x-ray pulses.

We calculated the evolution of the electron system in solid-density matter irradiated by high-intensity x-ray pulses between 2 and 8 keV using molecular dynamics. For pulses shorter than 40 fs, the kinetic energy distribution of the electrons is highly nonthermal during and right after the pulse, and a large fraction of the absorbed x-ray energy resides with the fast photoelectrons which equilibrate on the timescale of the pulse length. The average ionization and electron temperature of the bulk of the electrons are significantly lower than their equilibrium values.

Photoelectron dynamics in x-ray free-electron-laser diffractive imaging of biological samples.

X-ray free electron lasers hold the promise of enabling atomic-resolution diffractive imaging of single biological molecules. We develop a hybrid continuum-particle model to describe the x-ray induced damage and find that the photoelectron dynamics and electrostatic confinement strongly affect the time scale of the damage processes. These phenomena are not fully captured in hydrodynamic modeling approaches.

Atomic inner-shell X-ray laser at 1.46 nanometres pumped by an X-ray free-electron laser.

Since the invention of the laser more than 50 years ago, scientists have striven to achieve amplification on atomic transitions of increasingly shorter wavelength. The introduction of X-ray free-electron lasers makes it possible to pump new atomic X-ray lasers with ultrashort pulse duration, extreme spectral brightness and full temporal coherence. Here we describe the implementation of an X-ray laser in the kiloelectronvolt energy regime, based on atomic population inversion and driven by rapid K-shell photo-ionization using pulses from an X-ray free-electron laser. We established a population inversion of the Kα transition in singly ionized neon at 1.46 nanometres (corresponding to a photon energy of 849 electronvolts) in an elongated plasma column created by irradiation of a gas medium. We observed strong amplified spontaneous emission from the end of the excited plasma. This resulted in femtosecond-duration, high-intensity X-ray pulses of much shorter wavelength and greater brilliance than achieved with previous atomic X-ray lasers. Moreover, this scheme provides greatly increased wavelength stability, monochromaticity and improved temporal coherence by comparison with present-day X-ray free-electron lasers. The atomic X-ray lasers realized here may be useful for high-resolution spectroscopy and nonlinear X-ray studies.

Single mimivirus particles intercepted and imaged with an X-ray laser.

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

Near-ultraviolet luminescence of N2 irradiated by short X-ray pulses.

The Linac Coherent Light Source is an x-ray free-electron laser that recently demonstrated lasing in the 1.5-15 Å wavelength range. We report on luminescence measurements of a molecular nitrogen gas irradiated by ∼2 mJ, 80 fs x-ray pulses at energies of 0.83, 2.7, and 8.3 keV. These results provide a direct test of our current understanding of photoabsorption, electron dynamics, and fluorescence processes for such intense, ultrashort x-ray pulses. At 0.83 keV, the duration of the fluorescence signal depends strongly on space-charge effects. At 8.3 keV, space-charge effects are weak, and the signal duration is determined by the Auger electron dynamics.

Sacrificial tamper slows down sample explosion in FLASH diffraction experiments.

Intense and ultrashort x-ray pulses from free-electron lasers open up the possibility for near-atomic resolution imaging without the need for crystallization. Such experiments require high photon fluences and pulses shorter than the time to destroy the sample. We describe results with a new femtosecond pump-probe diffraction technique employing coherent 0.1 keV x rays from the FLASH soft x-ray free-electron laser. We show that the lifetime of a nanostructured sample can be extended to several picoseconds by a tamper layer to dampen and quench the sample explosion, making <1 nm resolution imaging feasible.

Molecular dynamics simulations of electron-ion temperature equilibration in an SF6 plasma.

We use classical molecular dynamics to investigate electron-ion temperature equilibration in a two-temperature SF6 plasma. We choose a density of 1.0 x 10;{19}SF_{6} molecules per cm;{3} and initial temperatures of T_{e} = 100 eV and T_{S} = T_{F} = 15 eV, in accordance with experiments currently underway at Los Alamos National Laboratory. Our computed relaxation time lies between two oft-used variants of the Landau-Spitzer relaxation formula which invoke static screening. Discrepancies are also found when comparing to the predictions made by more recent theoretical approaches. These differences should be large enough to be measured in the upcoming experiments.

Modeling of the damage dynamics of nanospheres exposed to x-ray free-electron-laser radiation.

Atomic-resolution diffraction imaging of biological particles using x-ray free-electron lasers (XFELs) at 1 A wavelength requires a detailed understanding of the photon-induced damage processes. We discuss how several aspects of existing continuum damage models can be tested during early operation of XFELs at lower x-ray energies in the range of 0.8-5 keV and low fluences, focusing particularly on macroscopic collective effects such as particle charging, expansion, and average ionization of nanospheres.

Effect of the coherence properties of self-amplified-spontaneous-emission x-ray free electron lasers on single-particle diffractive imaging.

The longitudinal coherence properties of self-amplified-spontaneous- emission x-ray free electron lasers limit the resolution of single-particle diffraction imaging. We found that for the Linac Coherent Light Source (LCLS) at a wavelength of 1.5 A the particles have to be smaller than 500 nm in diameter to achieve atomic-resolution imaging with a resolution length of less than 2 A, suggesting that the longitudinal coherence is sufficient for imaging most biomolecular samples of interest.

Camera for coherent diffractive imaging and holography with a soft-x-ray free-electron laser.

We describe a camera to record coherent scattering patterns with a soft-x-ray free-electron laser (FEL). The camera consists of a laterally graded multilayer mirror, which reflects the diffraction pattern onto a CCD detector. The mirror acts as a bandpass filter for both the wavelength and the angle, which isolates the desired scattering pattern from nonsample scattering or incoherent emission from the sample. The mirror also solves the particular problem of the extreme intensity of the FEL pulses, which are focused to greater than 10(14) W/cm2. The strong undiffracted pulse passes through a hole in the mirror and propagates onto a beam dump at a distance behind the instrument rather than interacting with a beam stop placed near the CCD. The camera concept is extendable for the full range of the fundamental wavelength of the free electron laser in Hamburg (FLASH) FEL (i.e., between 6 and 60 nm) and into the water window. We have fabricated and tested various multilayer mirrors for wavelengths of 32, 16, 13.5, and 4.5 nm. At the shorter wavelengths mirror roughness must be minimized to reduce scattering from the mirror. We have recorded over 30,000 diffraction patterns at the FLASH FEL with no observable mirror damage or degradation of performance.