Daewoong Nam

A three-dimensional movie of structural changes in bacteriorhodopsin

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science, this issue p. 1552 Time-resolved serial crystallography using an x-ray free electron laser reveals structural changes in bacteriorhodopsin. Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

Single-shot three-dimensional structure determination of nanocrystals with femtosecond X-ray free-electron laser pulses

Conventional three-dimensional (3D) structure determination methods require either multiple measurements at different sample orientations or a collection of serial sections through a sample. Here we report the experimental demonstration of single-shot 3D structure determination of an object; in this case, individual gold nanocrystals at ~5.5 nm resolution using ~10 fs X-ray free-electron laser pulses. Coherent diffraction patterns are collected from high-index-faceted nanocrystals, each struck by an X-ray free-electron laser pulse. Taking advantage of the symmetry of the nanocrystal and the curvature of the Ewald sphere, we reconstruct the 3D structure of each nanocrystal from a single-shot diffraction pattern. By averaging a sufficient number of identical nanocrystals, this method may be used to determine the 3D structure of nanocrystals at atomic resolution. As symmetry exists in many virus particles, this method may also be applied to 3D structure studies of such particles at nanometer resolution on femtosecond time scales.

Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells

Since its first experimental demonstration in 1999, coherent diffractive imaging (CDI) has been applied to image a broad range of samples using advanced synchrotron radiation, X-ray free-electron lasers, high harmonic generation and electrons. Here, the first experimental demonstration of cryogenic CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells is reported. As a proof of principle, the three-dimensional mass density of the sub-cellular organization of a Neospora caninum cell is determined based on its natural contrast.

Macromolecular structures probed by combining single-shot free-electron laser diffraction with synchrotron coherent X-ray imaging

Nanostructures formed from biological macromolecular complexes utilizing the self-assembly properties of smaller building blocks such as DNA and RNA hold promise for many applications, including sensing and drug delivery. New tools are required for their structural characterization. Intense, femtosecond X-ray pulses from X-ray free-electron lasers enable single-shot imaging allowing for instantaneous views of nanostructures at ambient temperatures. When combined judiciously with synchrotron X-rays of a complimentary nature, suitable for observing steady-state features, it is possible to perform ab initio structural investigation. Here we demonstrate a successful combination of femtosecond X-ray single-shot diffraction with an X-ray free-electron laser and coherent diffraction imaging with synchrotron X-rays to provide an insight into the nanostructure formation of a biological macromolecular complex: RNA interference microsponges. This newly introduced multimodal analysis with coherent X-rays can be applied to unveil nano-scale structural motifs from functional nanomaterials or biological nanocomplexes, without requiring a priori knowledge.

Multiple application X-ray imaging chamber for single-shot diffraction experiments with femtosecond X-ray laser pulses

X-ray free-electron lasers (XFELs) provide intense (∼1012 photons per pulse) coherent X-rays with ultra-short (∼10−14 s) pulse lengths. X-rays of such an unprecedented nature have introduced new means of atomic scale structural investigations, and discoveries are still ongoing. Effective use of XFELs would be further accelerated on a highly adaptable platform where most of the new experiments can be realized. Introduced here is the multiple-application X-ray imaging chamber (MAXIC), which is able to carry out various single-pulse diffraction experiments including single-shot imaging, nanocrystallographic data acquisition and ultra-fast pump–probe scattering for specimens in solid, liquid and gas phases. The MAXIC established at the SPring-8 angstrom compact free-electron laser (SACLA) has demonstrated successful applications in the aforementioned experiments, but is not limited to them. Also introduced are recent experiments on single-shot diffraction imaging of Au nanoparticles and serial crystallographic data collection of lysozyme crystals at SACLA.

Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.

Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency.

Development of an adaptable coherent x-ray diffraction microscope with the emphasis on imaging hydrated specimens.

This paper describes the development of a versatile coherent x-ray diffraction microscope capable of imaging biological specimens in solution. The microscope is a flexible platform accommodating various conditions, from low vacuum (10(-2) Pa) to helium gas filled ambient pressure. This flexibility greatly expands the application area, from in situ materials science to biology systems in their native state, by significantly relaxing restrictions to the sample environment. The coherent diffraction microscope has been used successfully to image a yeast cell immersed in buffer solution. We believe that the design of this coherent diffraction microscope can be directly adapted to various platforms such as table top soft x-ray laser, synchrotron x-ray sources, and x-ray free electron laser with minor relevant adjustments.

Three-dimensional reconstruction for coherent diffraction patterns obtained by XFEL

The developed reconstruction method can successfully identify the orientations of coherent X-ray diffraction patterns of an aerosol nanoparticle.

Fixed target single-shot imaging of nanostructures using thin solid membranes at SACLA

Single-shot imaging using femtosecond x-ray pulses from x-ray free electron lasers (XFELs) has revealed high-resolution structures of Au nanoparticles and biological macromolecular complexes. The x-ray pulse power of 10 GW is enough to vaporize specimen when it is focused on a few microns area. This makes it essential to have a single particle loader to provide fresh samples to the x-ray pulses. In this paper we introduce single-shot imaging at Spring-8 Angstrom Compact Free Electron Laser (SACLA) to investigate various types of specimen, from metallic nanoparticles to biological macromolecules, prepared on Si3N4 membrane. A significant reduction in sample consumption is achieved while maintaining a data acquisition rate of 30 Hz, which is compatible with the current SACLA operation rate.