A. Miahnahri

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.

Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature.

One Protein, Two Probes A central challenge in the use of x-ray diffraction to characterize macromolecular structure is the propensity of the high-energy radiation to damage the sample during data collection. Recently, a powerful accelerator-based, ultrafast x-ray laser source has been used to determine the geometric structures of small protein crystals too fragile for conventional diffraction techniques. Kern et al. (p. 491, published online 14 February) now pair this method with concurrent x-ray emission spectroscopy to probe electronic structure, as well as geometry, and were able to characterize the metal oxidation states in the oxygen-evolving complex within photosystem II crystals, while simultaneously verifying the surrounding protein structure. A powerful x-ray laser source can extract the geometry and electronic structure of metalloenzymes prior to damaging them. Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD-XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.

Room temperature femtosecond X-ray diffraction of photosystem II microcrystals

Most of the dioxygen on earth is generated by the oxidation of water by photosystem II (PS II) using light from the sun. This light-driven, four-photon reaction is catalyzed by the Mn4CaO5 cluster located at the lumenal side of PS II. Various X-ray studies have been carried out at cryogenic temperatures to understand the intermediate steps involved in the water oxidation mechanism. However, the necessity for collecting data at room temperature, especially for studying the transient steps during the O–O bond formation, requires the development of new methodologies. In this paper we report room temperature X-ray diffraction data of PS II microcrystals obtained using ultrashort (< 50 fs) 9 keV X-ray pulses from a hard X-ray free electron laser, namely the Linac Coherent Light Source. The results presented here demonstrate that the ”probe before destroy” approach using an X-ray free electron laser works even for the highly-sensitive Mn4CaO5 cluster in PS II at room temperature. We show that these data are comparable to those obtained in synchrotron radiation studies as seen by the similarities in the overall structure of the helices, the protein subunits and the location of the various cofactors. This work is, therefore, an important step toward future studies for resolving the structure of the Mn4CaO5 cluster without any damage at room temperature, and of the reaction intermediates of PS II during O–O bond formation.

Nanoflow electrospinning serial femtosecond crystallography

An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 µl min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min(-1) and diffracted to beyond 4 Å resolution, producing 14,000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.

Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers

X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode

The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this “probe-before-destroy” approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kβ1,3 XES spectra of MnII and Mn2III,IV complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.

High-intensity double-pulse X-ray free-electron laser.

The X-ray free-electron laser has opened a new era for photon science, improving the X-ray brightness by ten orders of magnitude over previously available sources. Similar to an optical laser, the spectral and temporal structure of the radiation pulses can be tailored to the specific needs of many experiments by accurately manipulating the lasing medium, that is, the electron beam. Here we report the generation of mJ-level two-colour hard X-ray pulses of few femtoseconds duration with an XFEL driven by twin electron bunches at the Linac Coherent Light Source. This performance represents an improvement of over an order of magnitude in peak power over state-of-the-art two-colour XFELs. The unprecedented intensity and temporal coherence of this new two-colour X-ray free-electron laser enable an entirely new set of scientific applications, ranging from X-ray pump/X-ray probe experiments to the imaging of complex biological samples with multiple wavelength anomalous dispersion.

Optical laser systems at the Linac Coherent Light Source

This manuscript serves as a reference to describe the optical laser sources and capabilities at the Linac Coherent Light Source.