Biomolecular imaging and electronic damage using X-ray free-electron lasers

Proposals to determine biomolecular structures from diffraction experiments using femtosecond X-ray free-electron laser (XFEL) pulses involve a conflict between the incident brightness required to achieve diffraction-limited atomic resolution and the electronic and structural damage induced by the illumination. Here we show that previous estimates of the conditions under which biomolecular structures may be obtained in this manner are unduly restrictive, because they are based on a coherent diffraction model that is not appropriate to the proposed interaction conditions. A more detailed imaging model derived from optical coherence theory and quantum electrodynamics is shown to be far more tolerant of electronic damage. The nuclear density is employed as the principal descriptor of molecular structure. The foundations of the approach may also be used to characterize electrodynamical processes by performing scattering experiments on complex molecules of known structure.Comment: 16 pages, 2 figure

Whole-pattern fitting technique in serial femtosecond nanocrystallography

Serial femtosecond X-ray crystallography (SFX) has created new opportunities in the field of structural analysis of protein nanocrystals. The intensity and timescale characteristics of the X-ray free-electron laser sources used in SFX experiments necessitate the analysis of a large collection of individual crystals of variable shape and quality to ultimately solve a single, average crystal structure. Ensembles of crystals are commonly encountered in powder diffraction, but serial crystallography is different because each crystal is measured individually and can be oriented via indexing and merged into a three-dimensional data set, as is done for conventional crystallography data. In this way, serial femtosecond crystallography data lie in between conventional crystallography data and powder diffraction data, sharing features of both. The extremely small sizes of nanocrystals, as well as the possible imperfections of their crystallite structure, significantly affect the diffraction pattern and raise the question of how best to extract accurate structure-factor moduli from serial crystallography data. Here it is demonstrated that whole-pattern fitting techniques established for one-dimensional powder diffraction analysis can be feasibly extended to higher dimensions for the analysis of merged SFX diffraction data. It is shown that for very small crystals, whole-pattern fitting methods are more accurate than Monte Carlo integration methods that are currently used

Mapping the continuous reciprocal space intensity distribution of X-ray serial crystallography

Serial crystallography using X-ray free-electron lasers enables the collection of tens of thousands of measurements from an equal number of individual crystals, each of which can be smaller than 1 mmin size. This manuscript describesan alternativeway of handling diffraction data recorded by serial femtosecond crystallography, by mapping the diffracted intensities into three-dimensional reciprocal space rather than integrating each image in two dimensions as in theclassical approach. We call this procedure ‘three-dimensional merging’. This procedure retains information about asymmetry in Bragg peaks and diffracted intensities between Bragg spots. This intensity distribution can be used toextract reflection intensities for structure determination and opens up novel avenues for post-refinement, while observed intensity between Bragg peaks and peak asymmetry are of potential use in novel direct phasing strategies

Phasing coherently illuminated nanocrystals bounded by partial unit cells

With the use of highly coherent femtosecond X-ray pulses from a free-electron laser, it is possible to record protein nanocrystal diffraction patterns with far more information than is present in conventional crystallographic diffractiondata. It has been suggested that diffraction phases may be retrieved from such data via iterative algorithms, without the use of a priori information and without restrictions on resolution. Here, we investigate the extension of thisapproach to nanocrystals with edge terminations that produce partial unit cells, and hence cannot be described by a common repeating unit cell. In this situation, the phase problemdescribed in previouswork must be reformulated.We demonstrate an approximate solution to this phase problem for crystals with random edge terminations

Nanocrystallography measurements of early stage synthetic malaria pigment

The recent availability of extremely intense, femtosecond X-ray free-electron laser (XFEL) sources has spurred the development of serial femtosecond nanocrystallography (SFX). Here, SFX is used to analyze nanoscale crystals of β-hematin, the synthetic form of hemozoin which is a waste by-product of the malaria parasite. This analysis reveals significant differences in β-hematin data collected during SFX and synchrotron crystallography experiments. To interpret these differences two possibilities are considered: structural differences between the nanocrystal and larger crystalline forms of β-hematin, and radiation damage. Simulation studies show that structural inhomogeneity appears at present to provide a better fit to the experimental data. If confirmed, these observations will have implications for designing compounds that inhibit hemozoin formation and suggest that, for some systems at least, additional information may be gained by comparing structures obtained from nanocrystals and macroscopic crystals of the same molecule

Measurements of long-range electronic correlations during femtosecond diffraction experiments performed on nanocrystals of buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered 'random'. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data

Nanocrystallography measurements of early stage synthetic malaria pigment

The recent availability of extremely intense, femtosecond X-ray free-electron laser (XFEL) sources has spurred the development of serial femtosecond nanocrystallography (SFX). Here, SFX is used to analyze nanoscale crystals of β-hematin, the synthetic form of hemozoin which is a waste by-product of the malaria parasite. This analysis reveals significant differences in β-hematin data collected during SFX and synchrotron crystallography experiments. To interpret these differences two possibilities are considered: structural differences between the nanocrystal and larger crystalline forms of β-hematin, and radiation damage. Simulation studies show that structural inhomogeneity appears at present to provide a better fit to the experimental data. If confirmed, these observations will have implications for designing compounds that inhibit hemozoin formation and suggest that, for some systems at least, additional information may be gained by comparing structures obtained from nanocrystals and macroscopic crystals of the same molecule