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Abstract: Copper-containing nitrite reductases (CuNiRs) are found in all three kingdoms of life and play a major role in the denitrification branch of the global nitro­gen cycle where nitrate is used in place of di­oxy­gen as an electron acceptor in respiratory energy metabolism. Several C- and N-terminal redox domain tethered CuNiRs have been identified and structurally characterized during the last decade. Our understanding of the role of tethered domains in these new classes of three-domain CuNiRs, where an extra cytochrome or cupredoxin domain is tethered to the catalytic two-domain CuNiRs, has remained limited. This is further compounded by a complete lack of substrate-bound structures for these tethered CuNiRs. There is still no substrate-bound structure for any of the as-isolated wild-type tethered enzymes. Here, structures of nitrite and product-bound states from a nitrite-soaked crystal of the N-terminal cupredoxin-tethered enzyme from the Hyphomicrobium denitrificans strain 1NES1 (Hd1NES1NiR) are provided. These, together with the as-isolated structure of the same species, provide clear evidence for the role of the N-terminal peptide bearing the conserved His27 in water-mediated anchoring of the substrate at the catalytic T2Cu site. Our data indicate a more complex role of tethering.

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Abstract: X-ray imaging of soft materials is often difficult because of the low contrast of the components. This particularly applies to frozen hydrated biological cells where the feature of interest can have a similar density to the surroundings. As a consequence, a high dose is often required to achieve the desired resolution. However, the maximum dose that a specimen can tolerate is limited by radiation damage. Results from 3D coherent diffraction imaging (CDI) of frozen hydrated specimens have given resolutions of ∼80 nm compared with the expected resolution of 10 nm predicted from theoretical considerations for identifying a protein embedded in water. Possible explanations for this include the inapplicability of the dose-fractionation theorem, the difficulty of phase determination, an overall object-size dependence on the required fluence and dose, a low contrast within the biological cell, insufficient exposure, and a variety of practical difficulties such as scattering from surrounding material. A recent article [Villaneuva-Perez et al. (2018), Optica, 5, 450–457] concluded that imaging by Compton scattering gave a large dose advantage compared with CDI because of the object-size dependence for CDI. An object-size dependence would severely limit the applicability of CDI and perhaps related coherence-based methods for structural studies. This article specifically includes the overall object size in the analysis of the fluence and dose requirements for coherent imaging in order to investigate whether there is a dependence on object size. The applicability of the dose-fractionation theorem is also discussed. The analysis is extended to absorption-based imaging and imaging by incoherent scattering (Compton) and fluorescence. This article includes analysis of the dose required for imaging specific low-contrast cellular organelles as well as for protein against water. This article concludes that for both absorption-based and coherent diffraction imaging, the dose-fractionation theorem applies and the required dose is independent of the overall size of the object. For incoherent-imaging methods such as Compton scattering, the required dose depends on the X-ray path length through the specimen. For all three types of imaging, the dependence of fluence and dose on a resolution d goes as 1/d4 when imaging uniform-density voxels. The independence of CDI on object size means that there is no advantage for Compton scattering over coherent-based imaging methods. The most optimistic estimate of achievable resolution is 3 nm for imaging protein molecules in water/ice using lensless imaging methods in the water window. However, the attainable resolution depends on a variety of assumptions including the model for radiation damage as a function of resolution, the efficiency of any phase-retrieval process, the actual contrast of the feature of interest within the cell and the definition of resolution itself. There is insufficient observational information available regarding the most appropriate model for radiation damage in frozen hydrated biological material. It is advocated that, in order to compare theory with experiment, standard methods of reporting results covering parameters such as the feature examined (e.g. which cellular organelle), resolution, contrast, depth of the material (for 2D), estimate of noise and dose should be adopted.

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Abstract: Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.

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Abstract: With the increasing trend of using microcrystals and intense microbeams at synchrotron X-ray beamlines, radiation damage becomes a more pressing problem. Theoretical calculations show that the photoelectrons that primarily cause damage can escape microcrystals. This effect would become more pronounced with decreasing crystal size as well as at higher energies. To prove this effect, data from cryocooled lysozyme crystals of dimensions 5 × 3 × 3 and 20 × 8 × 8 µm mounted on cryo-transmission electron microscopy (cryo-TEM) grids were collected at 13.5 and 20.1 keV using a PILATUS CdTe 2M detector, which has a similar quantum efficiency at both energies. Accurate absorbed doses were calculated through the direct measurement of individual crystal sizes using scanning electron microscopy after the experiment and characterization of the X-ray microbeam. The crystal lifetime was then quantified based on the D1/2 metric. In this first systematic study, a longer crystal lifetime for smaller crystals was observed and crystal lifetime increased at higher X-ray energies, supporting the theoretical predictions of photoelectron escape. The use of detector technologies specifically optimized for data collection at energies above 20 keV allows the theoretically predicted photoelectron escape to be quantified and exploited, guiding future beamline-design choices.

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Abstract: he application of thermoelectrics for energy harvesting depends strongly on operational reliability and it is therefore desirable to investigate the structural integrity of materials under operating conditions. We have developed an operando setup capable of simultaneously measuring X-ray scattering data and electrical resistance on pellets subjected to electrical current. Here, operando investigations of β-Zn4Sb3 are reported at current densities of 0.5, 1.14 and 2.3 A mm−2. At 0.5 A mm−2 no sample decomposition is observed, but Rietveld refinements reveal increased zinc occupancy from the anode to the cathode demonstrating zinc migration under applied current. At 1.14 A mm−2 β-Zn4Sb3 decomposes into ZnSb, but pair distribution function analysis shows that Zn2Sb2 units are preserved during the decomposition. This identifies the mobile zinc in β-Zn4Sb3 as the linkers between the Zn2Sb2 units. At 2.3 A mm−2 severe Joule heating triggers transition into the γ-Zn4Sb3 phase, which eventually decomposes into ZnSb, demonstrating Zn ion mobility also in γ-Zn4Sb3 under electrical current.