Damage-free Structures Research Articles

Damage-free structures of green copper nitrite reductase obtained by neutron crystallography and XFEL

Damage-free structures of green copper nitrite reductase obtained by neutron crystallography and XFEL

An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures.

Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br 2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br 2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.

Evolution of chemo-mechanical effects during single grit diamond scratching of monocrystalline silicon in the presence of potassium hydroxide

Efficiency and workability of silicon wafers for various applications depend on theirs surface integrity and damage free structures. Productivity of mechanical processing on silicon wafers is always limited by occurrence of cracks and brittle mode material removal at high depth of cut. This study presents a method of using controlled concentration of KOH solution during diamond micro-scratching of silicon and investigates the chemo-mechanical effects of KOH solution with respect to cutting speed. To establish the chemo-mechanical theory of cutting, responses of physical and material properties of silicon in presence of KOH solution are captured and correlated with mechanical responses during micro-scratching. Zeta potential and nanohardness are greatly reduced in KOH environment. Ductile mode is found to be extended by using 10 wt% KOH. Crack/chipping length is observed to be first increasing with increase in cutting speed at low range (0.6–20 mm/min) and then decreasing on further increase in the cutting speed (20–200 mm/min). Stability in cutting force signature and behavior of material flow along the scratch length is also found to be improved by using KOH. Adhesive wear and abrasive wear are found to be the main forms of single grit wear, which is significantly reduced in the presence of KOH. The experimental results on different mechanical and physical phenomena in this study are of great importance for establishing the theoretical insight into material removal behavior and selection of machining parameters during micro-machining of silicon wafer.

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

BackgroundThe invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. Scope of reviewRecent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. Major conclusionsXFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. General significanceXFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing ‘live’ protein molecules.

High-throughput structures of protein-ligand complexes at room temperature using serial femtosecond crystallography.

High-throughput X-ray crystal structures of protein-ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein-ligand complexes using SFX.

Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography.

Copper-containing nitrite reductases (CuNiRs) that convert NO2 - to NO via a CuCAT-His-Cys-CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that AspCAT and HisCAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D2O) is positioned with one deuteron directed towards AspCAT Oδ1 and one towards HisCAT N∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D2O molecule as opposed to D3O+ or OD-, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated AspCAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with AspCAT. Upon the binding of NO2 - a proton is transferred from the bridging water to the Oδ2 atom of AspCAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from AspCAT to the bound nitrite, enabling the reaction to proceed.

The expanding toolkit for structural biology: synchrotrons, X-ray lasers and cryoEM.

Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. The combination of results from X-ray free-electron lasers (XFELs), modern synchrotron crystallographic beamlines and cryo-electron microscopy (cryoEM) is proving to be particularly powerful. The highly brilliant undulator beamlines at modern synchrotron facilities have empowered the crystallographic revolution of high-throughput structure determination at high resolution. The brilliance of the X-rays at these crystallographic beamlines has enabled this to be achieved using microcrystals, but at the expense of an increased absorbed X-ray dose and a consequent vulnerability to radiation-induced changes. The advent of serial femtosecond crystallography (SFX) with X-ray free-electron lasers provides a new opportunity in which damage-free structures can be obtained from much smaller crystals (2 µm) and more complex macromolecules, including membrane proteins and multi-protein complexes. For redox enzymes, SFX provides a unique opportunity by providing damage-free structures at both cryogenic and ambient temperatures. The promise of being able to visualize macromolecular structures and complexes at high resolution without the need for crystals using X-rays has remained a dream, but recent technological advancements in cryoEM have made this come true and hardly a month goes by when the structure of a new/novel macromolecular assembly is not revealed. The uniqueness of cryoEM in providing structural information for multi-protein complexes, particularly membrane proteins, has been demonstrated by examples such as respirasomes. The synergistic use of cryoEM and crystallography in lead-compound optimization is highlighted by the example of the visualization of antimalarial compounds in cytochrome bc 1. In this short review, using some recent examples including our own work, we share the excitement of these powerful structural biology methods.

X-ray Free Electron Laser Radiation Damage through the S-State Cycle of the Oxygen-Evolving Complex of Photosystem II.

The oxygen-evolving complex (OEC) catalyzes water-splitting through a reaction mechanism that cycles the OEC through the "S-state" intermediates. Understanding structure/function relationsships of the S-states is crucial for elucidating the water-oxidation mechanism. Serial femtosecond X-ray crystallography has been used to obtain radiation damage-free structures. However, it remains to be established whether "diffraction-before-destruction" is actually accomplished or if significant changes are produced by the high-intensity X-ray pulses during the femtosecond scattering measurement. Here, we use ab initio molecular dynamics simulations to estimate the extent of structural changes induced on the femtosecond time scale. We found that the radiation damage is dependent on the bonding and charge of each atom in the OEC, in a manner that may provide lessons for XFEL studies of other metalloproteins. The maximum displacement of Mn and oxygen centers is 0.25 and 0.39 Å, respectively, during the 50 fs pulse, which is significantly smaller than the uncertainty given the 1.9 Å resolution of the current PSII crystal structures. However, these structural changes might be detectable when comparing isomorphous Fourier differences of electron density maps of the different S-states. One conclusion is that pulses shorter than 15 fs should be used to avoid significant radiation damage.

Monitoring Technical Conditions of Engineering Structures Using the Non-Linear Approach

Conventional methods of monitoring technical condition are based on detection of damage in the structures of buildings or facilities during the entire period of their operation. In spite of considerable interest displayed to this issue and a significant number of publications, there is no unity of opinions. These methods differ from each other in the sets of values fixed for investigations, the techniques of their recording, transfer and further processing. Today's rules and regulations for structural designs expand the scope of application of the structures operating in the elastic-plastic stage. These damage-free structures originally display the nonlinear properties and can be adequately described only by the non-linear models. This paper presents a method for determining the type and level of non-linearity from the structural oscillations data for monitoring the change in the health of structures. It is shown that a plot of acceleration against the magnitude of the displacement represents the restoring force of a structure. If the structure is damaged during a new striking motion, the phase trajectories in plane “acceleration-displacement” will deviate from its healthy signature.

高強度鋼を用いた構造システムの実大静的載荷試験による性能検証

This paper summarizes the results of static loading tests conducted for a full-scale, four-storied frame structure. The structure was composed of high-strength steel, H-SA700, and designed based on the separately proposed performance-based method intending damage-free structures even under extreme earthquakes. Loading history, in a form of enforced displacement at each story, was determined through response analyses. Test results showed that the structure would remain elastic under earthquakes of JMA seismic intensity 7, indicating the adequacy of the design method. Reusability of the structural components was also confirmed by visual and dimensional checking.

Mesh-free Modeling of Ultrasonic Wave Fields in Damaged Layered Half-spaces

Modeling of an ultrasonic wave field inside a layered half-space is carried out by using the mesh-free semi-analytical Distributed Point Source Method (DPSM). The complete field is computed in a layered half-space in presence and absence of defects. The layered structure is excited by a bounded ultrasonic beam generated by a finite-sized transducer. It is important to have theoretical models to predict the ultrasonic fields in damaged and damage-free structures for its nondestructive evaluation. Numerical exercises can be carried out aided by these theoretical models to determine the area of the most distorted ultrasonic field in presence of an internal anomaly. Several numerical examples are provided for an aluminum half-space attached to layers made of two different materials. The structure is excited by a bounded ultrasonic beam. Influence of the material properties and internal anomaly on the ultrasonic field pattern is demonstrated here.

Surface modification of InP by diffraction-patterning utilizing laser dry etching

Laser based dry etching of semiconductors offers a useful way of integrating patterning with growth for optoelectronic device development. In this article we demonstrate XeCl excimer laser based dry etching of InP. Experiments were carried out using a 10% gas mixture of chlorine diluted in helium. Studies were made of the effect of laser fluence on the etching process and how this influences pattern development. Based on these studies, surface electromagnetic waves were used to form ripple patterns and the optimum conditions for interference pattern development are reported. These studies show that a relatively low fluence is not conducive to pattern development. We also utilize diffraction from slits of different shapes in tandem with laser dry etching for the patterning of structures in semiconductors. This technique offers the potential to develop relatively damage-free structures. These structures may be suitable for devices used in a number of applications such as telecommunications.