Guided wave skew velocity correction in anisotropic laminates.

Guided ultrasonic wave propagation in anisotropic structures results in directional dependency of velocity and wave skewing effects that can impact the accuracy of damage detection. Phase and group velocities of the A0 guided wave mode, propagating in a unidirectional carbon fiber reinforced laminate, were investigated experimentally and through finite element analysis. A correction for the significant offset in phase and group velocities due to wave skewing effects is illustrated for both point and short line sources, achieving good agreement with theoretical calculations assuming planar wave fronts. The influence of the line excitation length on velocity measurements is discussed.

Post-acquisition mask misalignment correction for edge illumination x-ray phase contrast imaging.

Edge illumination x-ray phase contrast imaging uses a set of apertured masks to translate phase effects into variation of detected intensity. While the system is relatively robust against misalignment, mask movement during acquisition can lead to gradient artifacts. A method has been developed to correct the images by quantifying the misalignment post-acquisition and implementing correction maps to remove the gradient artifact. Images of a woven carbon fiber composite plate containing porosity were used as examples to demonstrate the image correction process. The gradient formed during image acquisition was removed without affecting the image quality, and results were subsequently used for quantification of porosity, indicating that the gradient correction did not affect the quantitative content of the images.

In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

AXSIS: Exploring the frontiers in attosecond X-ray science, imaging and spectroscopy.

X-ray crystallography is one of the main methods to determine atomic-resolution 3D images of the whole spectrum of molecules ranging from small inorganic clusters to large protein complexes consisting of hundred-thousands of atoms that constitute the macromolecular machinery of life. Life is not static, and unravelling the structure and dynamics of the most important reactions in chemistry and biology is essential to uncover their mechanism. Many of these reactions, including photosynthesis which drives our biosphere, are light induced and occur on ultrafast timescales. These have been studied with high time resolution primarily by optical spectroscopy, enabled by ultrafast laser technology, but they reduce the vast complexity of the process to a few reaction coordinates. In the AXSIS project at CFEL in Hamburg, funded by the European Research Council, we develop the new method of attosecond serial X-ray crystallography and spectroscopy, to give a full description of ultrafast processes atomically resolved in real space and on the electronic energy landscape, from co-measurement of X-ray and optical spectra, and X-ray diffraction. This technique will revolutionize our understanding of structure and function at the atomic and molecular level and thereby unravel fundamental processes in chemistry and biology like energy conversion processes. For that purpose, we develop a compact, fully coherent, THz-driven atto-second X-ray source based on coherent inverse Compton scattering off a free-electron crystal, to outrun radiation damage effects due to the necessary high X-ray irradiance required to acquire diffraction signals. This highly synergistic project starts from a completely clean slate rather than conforming to the specifications of a large free-electron laser (FEL) user facility, to optimize the entire instrumentation towards fundamental measurements of the mechanism of light absorption and excitation energy transfer. A multidisciplinary team formed by laser-, accelerator,- X-ray scientists as well as spectroscopists and biochemists optimizes X-ray pulse parameters, in tandem with sample delivery, crystal size, and advanced X-ray detectors. Ultimately, the new capability, attosecond serial X-ray crystallography and spectroscopy, will be applied to one of the most important problems in structural biology, which is to elucidate the dynamics of light reactions, electron transfer and protein structure in photosynthesis.

Evaluation of the 3D spatial distribution of the Calcium/Phosphorus ratio in bone using computed-tomography dual-energy analysis.

PURPOSE: The Calcium/Phosphorus (Ca/P) ratio was shown to vary between healthy bones and bones with osteoporotic symptoms. The relation of the Ca/P ratio to bone quality remains under investigation. To study this relation and determine if the ratio can be used to predict bone fractures, a non-invasive 3D imaging technique is required. The first aim of this study was to test the effectiveness of a computed-tomography dual-energy analysis (CT-DEA) technique developed to assess the Ca/P ratio in bone apatite (collagen-free bone) in identifying differences between healthy and inflammation-mediated osteoporotic (IMO) bones. The second aim was to extend the above technique for its application to a more complex structure, intact bone, that could potentially lead to clinical use. METHODS: For the first aim, healthy and IMO rabbit cortical bone apatite samples were assessed. For the second aim, some changes were made to the technique, which was applied to healthy and IMO intact bone samples. RESULTS: Statistically significant differences between healthy and IMO bone apatite were found for the bulk Ca/P ratio, low Ca/P ratio proportion and interconnected low Ca/P ratio proportion. For the intact bone samples, the bulk Ca/P ratio was found to be significantly different between healthy and IMO. CONCLUSIONS: Results show that the CT-DEA technique can be used to identify differences in the Ca/P ratio between healthy and osteoporotic, in both bone apatite and intact bone. With quantitative imaging becoming an increasingly important advancement in medical imaging, CT-DEA for bone decomposition could potentially have several applications.

A new technique for the assessment of the 3D spatial distribution of the calcium/phosphorus ratio in bone apatite.

The value and distribution of calcium/phosphorus (Ca/P) ratio in bone vary between healthy and osteoporotic bone. The purpose of this study was the development of a technique for the assessment of the 3D spatial distribution of Ca/P ratio in bone apatite, which could eventually be implemented through a conventional computed tomography (CT) system. A three-material mass-fraction decomposition CT dual energy analysis was optimized. The technique was validated using ten bone phantoms of different, known Ca/P ratio. Their measured average Ca/P ratio showed a mean/maximum deviation from the expected Ca/P ratio of 0.24/0.35. Additionally, three healthy and three inflammation-mediated osteoporotic (IMO) collagen-free rabbit tibia bone samples were assessed, providing promising preliminary results on real bone tissue. The average Ca/P ratios in all IMO samples (1.64-1.65) were found to be lower than in healthy samples (1.67-1.68). Osteoporotic regions in IMO samples were located using Ca/P ratio colour maps and Ca/P ratio values as low as 1.40 ± 0.26 were found. The low Ca/P ratio volume proportion in IMO samples (12.8%-13.9%) was found to be higher than in healthy (5.8%-8.3%) samples. A region growing technique showed a higher homogeneity of Ca/P ratio in healthy than in IMO bone samples.

Effect of amputation level on the stress transferred to the femur by an artificial limb directly attached to the bone.

Attachment of an artificial limb directly to the skeleton has a number of potential benefits and the technique has been implemented for several amputation sites. In this paper the transfer of stress from an external, transfemoral prosthesis to the femur during normal walking activity is investigated. The stress distribution in the femur and at the implant-bone interface is calculated using finite element analysis for the 3D geometry and inhomogeneous, anisotropic material properties obtained from a CT scan of a healthy femur. Attachment of the prosthetic leg at three different levels of amputation is considered. Stress concentrations are found at the distal end of the bone and adjacent to the implant tip and stress shielding is observed adjacent to the implant. It is found that the stress distribution in the femur distal to the epiphysis, where the femur geometry is close to cylindrical, can be predicted from a cylindrical finite element model, using the correct choice of bone diameter as measured from a radiograph. Proximal to the lesser trochanter the stress decreases as the femur geometry diverges significantly from a cylinder. The stress concentration at the distal, resected end of the bone is removed when a collared implant is employed. These findings form the basis for appropriate settings of an external fail-safe device to protect the bone from excessive stress in the event of an undue load.

X-ray diffraction from membrane protein nanocrystals.

Membrane proteins constitute > 30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is < 300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 µm. The results demonstrate that there are membrane protein crystals that contain < 100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain < 200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.

Powder diffraction from a continuous microjet of submicrometer protein crystals.

Atomic-resolution structures from small proteins have recently been determined from high-quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40, 133-143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129, 11865-11871]. While powder diffraction data have been obtained from batch samples of small crystal-suspensions, which are exposed to X-rays for long periods of time and undergo significant radiation damage, the proof-of-concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow-focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X-ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest-order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free-electron X-ray laser schemes, and for serial crystallography using a single-file beam of aligned hydrated molecules.

Dose, exposure time and resolution in serial X-ray crystallography.

The resolution of X-ray diffraction microscopy is limited by the maximum dose that can be delivered prior to sample damage. In the proposed serial crystallography method, the damage problem is addressed by distributing the total dose over many identical hydrated macromolecules running continuously in a single-file train across a continuous X-ray beam, and resolution is then limited only by the available molecular and X-ray fluxes and molecular alignment. Orientation of the diffracting molecules is achieved by laser alignment. The incident X-ray fluence (energy/area) is evaluated that is required to obtain a given resolution from (i) an analytical model, giving the count rate at the maximum scattering angle for a model protein, (ii) explicit simulation of diffraction patterns for a GroEL-GroES protein complex, and (iii) the spatial frequency cut-off of the transfer function following iterative solution of the phase problem, and reconstruction of an electron density map in the projection approximation. These calculations include counting shot noise and multiple starts of the phasing algorithm. The results indicate counting time and the number of proteins needed within the beam at any instant for a given resolution and X-ray flux. An inverse fourth-power dependence of exposure time on resolution is confirmed, with important implications for all coherent X-ray imaging. It is found that multiple single-file protein beams will be needed for sub-nanometer resolution on current third-generation synchrotrons, but not on fourth-generation designs, where reconstruction of secondary protein structure at a resolution of 7 A should be possible with relatively short exposures.

Diffraction and imaging from a beam of laser-aligned proteins: resolution limits.

The effect of the limited alignment of hydrated molecules is considered in a laser-aligned molecular beam, on diffraction patterns taken from the beam. Simulated patterns for a protein beam are inverted using the Fienup-Gerchberg-Saxton phasing algorithm, and the effect of limited alignment on the resolution of the resulting potential maps is studied. For a typical protein molecule (lysozyme) with anisotropic polarizability, it is found that up to 1 kW of continuous-wave near-infrared laser power (depending on dielectric constant), together with cooling to liquid-nitrogen temperatures, may be needed to produce sufficiently accurate alignment for direct observation of the secondary structure of proteins in the reconstructed potential or charge-density map. For a typical virus (TMV), a 50 W continuous-wave laser is adequate for subnanometre resolution at room temperature. The dependence of resolution on laser power, temperature, molecular size, shape and dielectric constant is analyzed.

Structure of photosystem I.

In plants and cyanobacteria, the primary step in oxygenic photosynthesis, the light induced charge separation, is driven by two large membrane intrinsic protein complexes, the photosystems I and II. Photosystem I catalyses the light driven electron transfer from plastocyanin/cytochrome c(6) on the lumenal side of the membrane to ferredoxin/flavodoxin at the stromal side by a chain of electron carriers. Photosystem I of Synechococcus elongatus consists of 12 protein subunits, 96 chlorophyll a molecules, 22 carotenoids, three [4Fe4S] clusters and two phylloquinones. Furthermore, it has been discovered that four lipids are intrinsic components of photosystem I. Photosystem I exists as a trimer in the native membrane with a molecular mass of 1068 kDa for the whole complex. The X-ray structure of photosystem I at a resolution of 2.5 A shows the location of the individual subunits and cofactors and provides new information on the protein-cofactor interactions. [P. Jordan, P. Fromme, H.T. Witt, O. Klukas, W. Saenger, N. Krauss, Nature 411 (2001) 909-917]. In this review, biochemical data and results of biophysical investigations are discussed with respect to the X-ray crystallographic structure in order to give an overview of the structure and function of this large membrane protein.

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: the tyrosine radical Y(D)(*).

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.

Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution.

Life on Earth depends on photosynthesis, the conversion of light energy from the Sun to chemical energy. In plants, green algae and cyanobacteria, this process is driven by the cooperation of two large protein-cofactor complexes, photosystems I and II, which are located in the thylakoid photosynthetic membranes. The crystal structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus described here provides a picture at atomic detail of 12 protein subunits and 127 cofactors comprising 96 chlorophylls, 2 phylloquinones, 3 Fe4S4 clusters, 22 carotenoids, 4 lipids, a putative Ca2+ ion and 201 water molecules. The structural information on the proteins and cofactors and their interactions provides a basis for understanding how the high efficiency of photosystem I in light capturing and electron transfer is achieved.

Crystal structure of photosystem II from Synechococcus elongatus at 3.8 A resolution.

Oxygenic photosynthesis is the principal energy converter on earth. It is driven by photosystems I and II, two large protein-cofactor complexes located in the thylakoid membrane and acting in series. In photosystem II, water is oxidized; this event provides the overall process with the necessary electrons and protons, and the atmosphere with oxygen. To date, structural information on the architecture of the complex has been provided by electron microscopy of intact, active photosystem II at 15-30 A resolution, and by electron crystallography on two-dimensional crystals of D1-D2-CP47 photosystem II fragments without water oxidizing activity at 8 A resolution. Here we describe the X-ray structure of photosystem II on the basis of crystals fully active in water oxidation. The structure shows how protein subunits and cofactors are spatially organized. The larger subunits are assigned and the locations and orientations of the cofactors are defined. We also provide new information on the position, size and shape of the manganese cluster, which catalyzes water oxidation.

In vitro reconstitution and biophysical characterization of OEP16, an outer envelope pore protein of pea chloroplasts.

More than 30% of all proteins in the living cell are membrane proteins; most of them occur in the native membranes only in very low amounts, which hinders their functional and structural investigation. Here we describe the in vitro reconstitution of overexpressed Outer Envelope Protein 16 (OEP16) from pea chloroplasts, a cation-selective channel, which has been purified from E. coli inclusion bodies. Reconstitution in detergent micelles was monitored by CD and fluorescence spectroscopy. Electron microscopy showed a homogeneous size distribution of the reconstituted protein, and differential scanning calorimetry gave an estimate of the enthalpy of protein folding. First protein crystals were obtained that have to be further refined for X-ray structural analysis. The described methods of membrane protein reconstitution and biophysical analysis might prove helpful in the study of other membrane proteins.

First photosystem II crystals capable of water oxidation.

Oxygen evolution and proton release of crystallised photosystem II core complexes isolated from Synechococcus elongatus were measured. The yields show that the crystals themselves are capable of highly active water oxidation. This opens the possibility for the structural analysis of the outstanding water-oxidising apparatus.

Photosystem I, an improved model of the stromal subunits PsaC, PsaD, and PsaE.

An improved electron density map of photosystem I (PSI) calculated at 4-A resolution yields a more detailed structural model of the stromal subunits PsaC, PsaD, and PsaE than previously reported. The NMR structure of the subunit PsaE of PSI from Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062) has been used as a model to interpret the region of the electron density map corresponding to this subunit. The spatial orientation with respect to other subunits is described as well as the possible interactions between the stromal subunits. A first model of PsaD consisting of a four-stranded beta-sheet and an alpha-helix is suggested, indicating that this subunit partly shields PsaC from the stromal side. In addition to the improvements on the stromal subunits, the structural model of the membrane-integral region of PSI is also extended. The current electron density map allows the identification of the N and C termini of the subunits PsaA and PsaB. The 11-transmembrane alpha-helices of these subunits can now be assigned uniquely to the hydrophobic segments identified by hydrophobicity analyses.

Localization of two phylloquinones, QK and QK', in an improved electron density map of photosystem I at 4-A resolution.

An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-A resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques.