Keith O. Hodgson

Femtosecond diffractive imaging with a soft-X-ray free-electron laser

Theory predicts1,2,3,4 that, with an ultrashort and extremely bright coherent X-ray pulse, a single diffraction pattern may be recorded from a large macromolecule, a virus or a cell before the sample explodes and turns into a plasma. Here we report the first experimental demonstration of this principle using the FLASH soft-X-ray free-electron laser. An intense 25 fs, 4×1013 W cm−2 pulse, containing 1012 photons at 32 nm wavelength, produced a coherent diffraction pattern from a nanostructured non-periodic object, before destroying it at 60,000 K. A novel X-ray camera assured single-photon detection sensitivity by filtering out parasitic scattering and plasma radiation. The reconstructed image, obtained directly from the coherent pattern by phase retrieval through oversampling5,6,7,8,9, shows no measurable damage, and is reconstructed at the diffraction-limited resolution. A three-dimensional data set may be assembled from such images when copies of a reproducible sample are exposed to the beam one by one10.

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.

Structural Genomics of the Thermotoga maritima Proteome Implemented in a High-throughput Structure Determination Pipeline

Structural genomics is emerging as a principal approach to define protein structure–function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Use of one‐electron theory for the interpretation of near edge structure in K‐shell x‐ray absorption spectra of transition metal complexes

We report the results of multiple scattered wave SCF X‐alpha calculations of the one‐electron cross section for K‐shell photoabsorption in the molecular complexes MoO4−−, CrO4−−, and MoS4−−. We show that the method can successfully account for energy separations and relative cross sections of spectral features both below and above the K‐shell ionization threshold. Furthermore, we show: (a) that the first fairly intense peak on the low energy side of the rising edge for molybdate and chromate is due to a dipole allowed transition to a bound antibonding state of mainly nd character on the metal ion; this transition is possible because of the mixing with the ligand p orbitals having the proper T2 symmetry induced by the tetrahedral molecular potential; (b) the shoulder on the rising absorption edge can be explained by the beginning of the steplike continuum absorption when convolved with a Lorentzian function of frequency to imitate lifetime and monochromator broadening: (c) the main absorption peak is due t...

A codeposition route to CuI-pyridine coordination complexes for organic light-emitting diodes.

We demonstrate a new approach for utilizing CuI coordination complexes as emissive layers in organic light-emitting diodes that involves in situ codeposition of CuI and 3,5-bis(carbazol-9-yl)pyridine (mCPy). With a simple three-layer device structure, pure green electroluminescence at 530 nm from a Cu(I) complex was observed. A maximum luminance and external quantum efficiency (EQE) of 9700 cd/m(2) and 4.4%, respectively, were achieved. The luminescent species was identified as [CuI(mCPy)(2)](2) on the basis of photophysical studies of model complexes and X-ray absorption spectroscopy.

Structure and reactivity of a mononuclear non-haem iron( III )–peroxo complex

Oxygen-containing mononuclear iron species—iron(iii)–peroxo, iron(iii)–hydroperoxo and iron(iv)–oxo—are key intermediates in the catalytic activation of dioxygen by iron-containing metalloenzymes. It has been difficult to generate synthetic analogues of these three active iron–oxygen species in identical host complexes, which is necessary to elucidate changes to the structure of the iron centre during catalysis and the factors that control their chemical reactivities with substrates. Here we report the high-resolution crystal structure of a mononuclear non-haem side-on iron(iii)–peroxo complex, [Fe(iii)(TMC)(OO)]+. We also report a series of chemical reactions in which this iron(iii)–peroxo complex is cleanly converted to the iron(iii)–hydroperoxo complex, [Fe(iii)(TMC)(OOH)]2+, via a short-lived intermediate on protonation. This iron(iii)–hydroperoxo complex then cleanly converts to the ferryl complex, [Fe(iv)(TMC)(O)]2+, via homolytic O–O bond cleavage of the iron(iii)–hydroperoxo species. All three of these iron species—the three most biologically relevant iron–oxygen intermediates—have been spectroscopically characterized; we note that they have been obtained using a simple macrocyclic ligand. We have performed relative reactivity studies on these three iron species which reveal that the iron(iii)–hydroperoxo complex is the most reactive of the three in the deformylation of aldehydes and that it has a similar reactivity to the iron(iv)–oxo complex in C–H bond activation of alkylaromatics. These reactivity results demonstrate that iron(iii)–hydroperoxo species are viable oxidants in both nucleophilic and electrophilic reactions by iron-containing enzymes.

Imaging whole Escherichia coli bacteria by using single-particle x-ray diffraction

We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 Å. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining single-particle diffraction with x-ray free electron lasers.

Structural characterization of horseradish peroxidase using EXAFS spectroscopy. Evidence for Fe = O ligation in compounds I and II

Extended X-ray absorption fine structure spectroscopy has been utilized to determine the structural environment of the heme iron sites in horseradish peroxidase compounds I and II. For comparison, analogous studies have been undertaken on putative ferryl (Fe/sup IV/=O) porphyrin model compounds and on crystallographically characterized Cr/sup IV/=O and Cr/sup V/ identical with N porphyrins. In a preliminary communication, they suggested that a short ca. 1.6 A Fe-O bond is present in the high valent forms of both the enzyme and the synthetic porphyrins. The present work demonstrates unambiguously that a short, ca. 1.64 A, Fe-O bond length is present both in HRP compounds I and II and in their synthetic analogues. This structure is consistent only with an oxo-ferryl (Fe=O) complex as the active oxygen species in horseradish peroxidase. The structural details, their implications for heme protein mediated oxygen activation, and the difference between their results and those recently published by other workers.

Observation of an electric quadrupole transition in the X-ray absorption spectrum of a Cu(II) complex

Abstract The polarized X-ray absorption cross section of the ls → 3d transition in a square planar CuCl2−4 complex has been measured with respect to rotation about an axis normal to the CuCl4. The cross section exhibits four-fold periodicity about this axis, indicating that the transition is primarily due to coupling with the electric quadrupole component of the radiation. The vibronically allowed dipole transition is approximately one-third as the quadrupolar cross section. These observations are in agreement with SCF Xα multiple-scattered wave calculations, and may have implications for the intepretation of other X-ray absorption spectra. The half-filled d orbital is shown to have the angular characteristics of dx2−y2.

Solvent Tuning of Electrochemical Potentials in the Active Sites of HiPIP Versus Ferredoxin

A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.