From solutions of prehnitene and the ternary halides (SnCl)[MCl4] (M = Al, Ga) in chloro­benzene, the new cationic SnII–π-arene complexes catena-poly[[chlorido­aluminate(III)]-tri-μ-chlorido-4':1κ2Cl,1:2κ4Cl-[(η6-1,2,3,4-tetra­meth­yl­benzene)­tin(II)]-di-μ-chlorido-2:3κ4Cl-[(η6-1,2,3,4-tetra­methyl­benzene)­tin(II)]-di-μ-chlorido-3:4κ4Cl-[chlorido­aluminate(III)]-μ-chlorido-4:1'κ2Cl], [Al2Sn2Cl10(C10H14)2]n, (1) and catena-poly[[chlorido­gallate(III)]-tri-μ-chlor­ido-4':1κ2Cl,1:2κ4Cl-[(η6-1,2,3,4-tetra­methyl­benzene)­tin(II)]-di-μ-chlorido-2:3κ4Cl-[(η6-1,2,3,4-tetra­methyl­benzene)­tin(II)]-di-μ-chlorido-3:4κ4Cl-[chlor­ido­gallate(III)]-μ-chlorido-4:1'κ2Cl], [Ga2Sn2Cl10(C10H14)2]n, (2), were isolated. In these first main-group metal–prehnitene complexes, the distorted η6 arene π-bonding to the tin atoms of the Sn2Cl22+ moieties in the centre of [{1,2,3,4-(CH3)4C6H2}2Sn2Cl2][MCl4]2 repeating units (site symmetry overline{1}) is characterized by: (i) a significant ring slippage of ca 0.4 Å indicated by the dispersion of Sn—C distances [1: 2.881 (2)–3.216 (2) Å; 2: 2.891 (3)–3.214 (3) Å]; (ii) the non-methyl-substituted arene C atoms positioned closest to the SnII central atom; (iii) a pronounced tilt of the plane of the arene ligand against the plane of the central (Sn2Cl2)2+ four-membered ring species [1: 15.59 (11)°, 2: 15.69 (9)°]; (iv) metal–arene bonding of medium strength as illustrated by application of the bond-valence method in an indirect manner, defining the π-arene bonding inter­action of the SnII central atoms as s(SnII—arene) = 2 − Σs(SnII—Cl), that gives s(SnII—arene) = 0.37 and 0.38 valence units for the aluminate and the gallate, respectively, indicating that comparatively strong main-group metal–arene bonding is present and in line with the expectation that [AlCl4]− is the slightly weaker coordinating anion as compared to [GaCl4]−.

The title hydrated salt, tris­[hexa­amminecobalt(III)] tetraoxidorhenate(VII) tetra­kis­[hexa­fluorido­rhenate(IV)] hexa­hydrate, arose unexpectedly due to possible contamination of the K2ReF6 starting material with KReO4. It consists of octa­hedral [Co(NH3)6]3+ cation (Co1 site symmetry 1), tetra­hedral [ReVIIO4]− anions (Re site symmetry 1) and octa­hedral [ReIVF6]2− anions (Re site symmetries 1and overline{3}). The [ReF6]2− octa­hedral anions (mean Re—F = 1.834 Å), [Co(NH3)6]3+ octa­hedral cations (mean Co—N = 1.962 Å), and the [ReO4]− tetra­hedral anion (mean Re—O = 1.719 Å) are slightly distorted. A network of N—H⋯F hydrogen bonds consolidates the structure. The crystal studied was refined as a two-component twin.

Three new isotypic heteropolynuclear complexes, namely penta­aqua­carbonato­penta­kis­(glycinehydroxamato)nitrato­penta­copper(II)lanthanide(III) x-hydrate, [LnCu5(GlyHA)5(CO3)(NO3)(H2O)5]·xH2O (GlyHA2− is glycine­hydrox­amate, N-hy­droxy­glycinamidate or amino­aceto­hydroxamate, C2H4N2O22−), with lanthanide(III) (LnIII) = gadolinium (Gd, 1, x = 3.5), dysprosium (Dy, 2, x = 3.28) and holmium (Ho, 3, x = 3.445), within a 15-metallacrown-5 class were obtained on reaction of lanthanide(III) nitrate, copper(II) acetate and sodium glycinehydroxamate. Complexes 1–3 contain five copper(II) ions and five bridging GlyHA2− anions, forming a [CuGlyHA]5 metallamacrocyclic core. The LnIII ions are coordinated to the metallamacrocycle through five O-donor hydroxamates. The electroneutrality of complexes 1–3 is achieved by a bidentate carbonate anion coordinated to the LnIII ion and a monodentate nitrate anion coordinated apically to one of the copper(II) ions of the metallamacrocycle. The lattice parameters of complexes 1–3 are similar to those previously reported for an EuIII–CuII 15-metallacrown-5 complex with glycine­hydroxamate of proposed composition [EuCu5(GlyHA)5(OH)(NO3)2(H2O)4]·3.5H2O [Stemmler et al. (1999). Inorg. Chem. 38, 2807–2817]. High-quality X-ray data obtained for 1–3 have allowed a re-evaluation of the X-ray data solution proposed earlier for the EuCu5 complex and suggest that the formula is actually [EuCu5(GlyHA)5(CO3)(NO3)(H2O)5]·3.5H2O.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV–visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15–40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.

The disposition of functional groups can induce variations in the nature and type of interactions and hence affect the molecular recognition and self-assembly mechanism in cocrystals. To better understand the formation of cocrystals on a molecular level, the effects of disposition of functional groups on the formation of cocrystals were systematically and comprehensively investigated using cresol isomers (o-, m-, p-cresol) as model compounds. Consistency and variability in these cocrystals containing positional isomers were found and analyzed. The structures, molecular recognition and self-assembly mechanism of supramolecular synthons in solution and in their corresponding cocrystals were verified by a combined experimental and theoretical calculation approach. It was found that the heterosynthons (heterotrimer or heterodimer) combined with O—H⋯N hydrogen bonding played a significant role. Hirshfeld surface analysis and computed interaction energy values were used to determine the hierarchical ordering of the weak interactions. The quantitative analyses of charge transfers and molecular electrostatic potential were also applied to reveal and verify the reasons for consistency and variability. Finally, the molecular recognition, self-assembly and evolution process of the supramolecular synthons in solution were investigated. The results confirm that the supramolecular synthon structures formed initially in solution would be carried over to the final cocrystals, and the supramolecular synthon structures are the precursors of cocrystals and the information memory of the cocrystallization process, which is evidence for classical nucleation theory.

100 kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200 keV offers both cost savings and potentially improved imaging. The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage. For frozen hydrated specimens around 300 Å in thickness, the predicted optimal electron energy for imaging is 100 keV. Currently available electron cryomicroscopes in the 100–120 keV range are not optimized for cryoEM as they lack both the spatially coherent illumination needed for the high defocus used in cryoEM and imaging detectors optimized for 100 keV electrons. To demonstrate the potential of imaging at 100 kV, the voltage of a standard, commercial 200 kV field-emission gun (FEG) microscope was reduced to 100 kV and a side-entry cryoholder was used. As high-efficiency, large-area cameras are not currently available for 100 keV electrons, a commercial hybrid pixel camera designed for X-ray detection was attached to the camera chamber and was used for low-dose data collection. Using this configuration, five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein and haemoglobin, ranging in size from 4.5 MDa to 64 kDa with corresponding diameters from 320 to 72 Å. These five data sets were used to reconstruct 3D structures with resolutions between 8.4 and 3.4 Å. Based on this work, the practical advantages and current technological limitations to single-particle cryoEM at 100 keV are considered. These results are also discussed in the context of future microscope development towards the goal of rapid, simple and widely available structure determination of any purified biological specimen.

3D electron diffraction (3DED) has been used to follow polymorph evolution in the crystallization of glycine from aqueous solution. The three polymorphs of glycine which exist under ambient conditions follow the stability order β < α < γ. The least stable β polymorph forms within the first 3 min, but this begins to yield the α-form after only 1 min more. Both structures could be determined from continuous rotation electron diffraction data collected in less than 20 s on crystals of thickness ∼100 nm. Even though the γ-form is thermodynamically the most stable polymorph, kinetics favour the α-form, which dominates after prolonged standing. In the same sample, some β and one crystallite of the γ polymorph were also observed.

The crystal structure of ilmajokite, a rare Na-K-Ba-Ce-titanosilicate from the Khibiny mountains, Kola peninsula, Russia, has been solved using single-crystal X-ray diffraction data. The crystal structure is based on a 3D titanosilicate framework consisting of trigonal prismatic titanosilicate (TPTS) clusters centered by Ce3+ in [9]-coordination. Four adjacent TPTS clusters are linked into four-membered rings within the (010) plane and connected via ribbons parallel to 101. The ribbons are organized into layers parallel to (010) and modulated along the a axis with a modulation wavelength of csinβ = 32.91 Å and an amplitude of ∼b/2 = 13.89 Å. The layers are linked by additional silicate tetrahedra. Na+, K+, Ba2+ and H2O groups occur in the framework cavities and have different occupancies and coordination environments. The crystal structure of ilmajokite can be separated into eight hierarchical levels: atoms, coordination polyhedra, TPTS clusters, rings, ribbons, layers, the framework and the whole structure. The information-based analysis allows estimation of the complexity of the structure as 8.468 bits per atom and 11990.129 bits per cell. According to this analysis, ilmajokite is the third-most complex mineral known to date after ewingite and morrisonite, and is the most complex mineral framework structure, comparable in complexity to paulingite-(Ca) (11 590.532 bits per cell).

SMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc–ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc–ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.

Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times. By combining serial synchrotron crystallography (SSX) with 3D printing, a new experimental platform has been created that tackles these challenges. An affordable 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) is reported that allows data to be collected from protein microcrystals in a 3D flow with very high hit and indexing rates, while keeping the sample consumption low. The miniaturized 3D-MiXD can be rapidly installed into virtually any synchrotron beamline with only minimal adjustments. This efficient collection scheme in combination with its mixing geometry paves the way for recording molecular movies at synchrotrons by mixing-triggered millisecond time-resolved SSX.

Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.

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.

Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5', the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.

Nucleation of crystals from solution is fundamental to many natural and industrial processes. In this work, the molecular mechanism of conformational polymorphism nucleation and the links between the molecular conformation in solutions and in crystals were investigated in detail by using 5-nitro­furazone as the model compound. Different polymorphs were prepared, and the conformations in solutions obtained by dissolving different polymorphs were analysed and compared. The solutions of 5-nitro­furazone were proven to contain multiple conformers through quantum chemical computation, Raman spectra analysis, 2D nuclear Overhauser effect spectroscopy spectra analysis and molecular dynamics simulation. The conformational evolution and desolvation path was illustrated according to the 1H NMR spectra of solutions with different concentrations. Finally, based on all the above analysis, the molecular conformational evolution path during nucleation of 5-nitro­furazone was illustrated. The results presented in this work shed a new light on the molecular mechanism of conformational polymorphism nucleation in solution.

Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1–CD98hc, a HAT, transports essential amino acids and drugs across the blood–brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1–CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3–3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127–130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination.

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallo­graphy, were exploited to reach these conclusions and provide additional insights.

Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed β-propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a variety of triads with possible ion-binding characteristics lurking in OLF domains in invertebrate chordates such as Arthropoda (Asp–Glu–Ser), Nematoda (Asp–Asp–His) and Echinodermata (Asp–Glu–Lys). To test ion binding and to extend the observed connection between ion binding and distal structural rearrangements, consensus triads from these phyla were installed in the myoc-OLF. All three protein variants exhibit wild-type-like or better stability, but their calcium-binding properties differ, concomitant with new structural deviations from wild-type myoc-OLF. Taken together, the results indicate that calcium binding is not intrinsically destabilizing to myoc-OLF or required to observe a well ordered side helix, and that ion binding is a differential feature that may underlie the largely elusive biological function of OLF propellers.

Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportion­ation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

Atrazine is an s-triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s-triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Å resolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

Reducing the sample-exchange time is a crucial issue in maximizing the throughput of macromolecular crystallography (MX) beamlines because the diffraction data collection itself is completed within a minute in the era of pixel-array detectors. To this end, an upgraded sample changer, SPACE-II, has been developed on the basis of the previous model, SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), at the BL41XU beamline at SPring-8. SPACE-II achieves one sample-exchange step within 16 s, of which its action accounts for only 11 s, because of three features: (i) the implementation of twin arms that enable samples to be exchanged in one cycle of mount-arm action, (ii) the implementation of long-stroke mount arms that allow samples to be exchanged without withdrawal of the detector and (iii) the use of a fast-moving translation and rotation stage for the mount arms. By pre-holding the next sample prior to the sample-exchange sequence, the time was further decreased to 11 s in the case of automatic data collection, of which the action of SPACE-II accounted for 8 s. Moreover, the sample capacity was expanded from four to eight Uni-Pucks. The performance of SPACE-II has been demonstrated in over two years of operation at BL41XU; the average number of samples mounted on the diffractometer in one day was increased from 132 to 185, with an error rate of 0.089%, which counted incidents in which users could not continue with an experiment without recovery work by entering the experimental hutch. On the basis of these results, SPACE-II has been installed at three other MX beamlines at SPring-8 as of July 2019. The fast and highly reliable SPACE-II is now one of the most important pieces of infrastructure for the MX beamlines at SPring-8, providing users with the opportunity to fully make use of limited beamtime with brilliant X-rays.

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.

Image-processing software has always been an integral part of structure determination by cryogenic electron microscopy (cryo-EM). Recent advances in hardware and software are recognized as one of the key factors in the so-called cryo-EM resolution revolution. Increasing computational power has opened many possibilities to consider more demanding algorithms, which in turn allow more complex biological problems to be tackled. Moreover, data processing has become more accessible to many experimental groups, with computations that used to last for many days at supercomputing facilities now being performed in hours on personal workstations. All of these advances, together with the rapid expansion of the community, continue to pose challenges and new demands on the software-development side. In this article, the development of emcore and emvis, two basic software libraries for image manipulation and data visualization in cryo-EM, is presented. The main goal is to provide basic functionality organized in modular components that other developers can reuse to implement new algorithms or build graphical applications. An additional aim is to showcase the importance of following established practices in software engineering, with the hope that this could be a first step towards a more standardized way of developing and distributing software in the field.

Electron diffraction tomography (EDT) has gained increasing interest, starting with the development of automated electron diffraction tomography (ADT) which enables the collection of three-dimensional electron diffraction data from nano-sized crystals suitable for ab initio structure analysis. A basic description of the ADT method, nowadays recognized as a reliable and established method, as well as its special features and general applicability to different transmission electron microscopes is provided. In addition, the usability of ADT for crystal structure analysis of single nano-sized crystals with and without special crystallographic features, such as twinning, modulations and disorder is demonstrated.

Propagation-based phase-contrast X-ray imaging is by now a well established imaging technique, which – as a full-field technique – is particularly useful for tomography applications. Since it can be implemented with synchrotron radiation and at laboratory micro-focus sources, it covers a wide range of applications. A limiting factor in its development has been the phase-retrieval step, which was often performed using methods with a limited regime of applicability, typically based on linearization. In this work, a much larger set of algorithms, which covers a wide range of cases (experimental parameters, objects and constraints), is compiled into a single toolbox – the HoloTomoToolbox – which is made publicly available. Importantly, the unified structure of the implemented phase-retrieval functions facilitates their use and performance test on different experimental data.

A lipid droplet (LD) core of a cell consists mainly of neutral lipids, triacylglycerols and/or steryl esters (SEs). The structuration of these lipids inside the core is still under debate. Lipid segregation inside LDs has been observed but is sometimes suggested to be an artefact of LD isolation and chemical fixation. LD imaging in their native state and in unaltered cellular environments appears essential to overcome these possible technical pitfalls. Here, imaging techniques for ultrastructural study of native LDs in cellulo are provided and it is shown that LDs are organized structures. Cryo soft X-ray tomography and deep-ultraviolet (DUV) transmittance imaging are showing a partitioning of SEs at the periphery of the LD core. Furthermore, DUV transmittance and tryptophan/tyrosine auto-fluorescence imaging on living cells are combined to obtain complementary information on cell chemical contents. This multimodal approach paves the way for a new label-free organelle imaging technique in living cells.

A scanning soft X-ray spectromicroscope was recently developed based mainly on the photon-in/photon-out measurement scheme for the investigation of local electronic structures on the surfaces and interfaces of advanced materials under conditions ranging from low vacuum to helium atmosphere. The apparatus was installed at the soft X-ray beamline (BL17SU) at SPring-8. The characteristic features of the apparatus are described in detail. The feasibility of this spectromicroscope was demonstrated using soft X-ray undulator radiation. Here, based on these results, element-specific two-dimensional mapping and micro-XAFS (X-ray absorption fine structure) measurements are reported, as well as the observation of magnetic domain structures from using a reference sample of permalloy micro-dot patterns fabricated on a silicon substrate, with modest spatial resolution (e.g. ∼500 nm). Then, the X-ray radiation dose for Nafion® near the fluorine K-edge is discussed as a typical example of material that is not radiation hardened against a focused X-ray beam, for near future experiments.

A dedicated X-ray imaging detector for 200 keV high-energy X-ray microtomography was developed to realize high-efficiency high-resolution imaging while keeping the field of view wide.

The Inorganic Crystal Structure Database (ICSD) is the world's largest database of fully evaluated and published crystal structure data, mostly obtained from experimental results. However, the purely experimental approach is no longer the only route to discover new compounds and structures. In the past few decades, numerous computational methods for simulating and predicting structures of inorganic solids have emerged, creating large numbers of theoretical crystal data. In order to take account of these new developments the scope of the ICSD was extended in 2017 to include theoretical structures which are published in peer-reviewed journals. Each theoretical structure has been carefully evaluated, and the resulting CIF has been extended and standardized. Furthermore, a first classification of theoretical data in the ICSD is presented, including additional categories used for comparison of experimental and theoretical information.

A sample-injection device has been developed at SPring-8 Angstrom Compact Free-Electron Laser (SACLA) for serial femtosecond crystallography (SFX) at atmospheric pressure. Microcrystals embedded in a highly viscous carrier are stably delivered from a capillary nozzle with the aid of a coaxial gas flow and a suction device. The cartridge-type sample reservoir is easily replaceable and facilitates sample reloading or exchange. The reservoir is positioned in a cooling jacket with a temperature-regulated water flow, which is useful to prevent drastic changes in the sample temperature during data collection. This work demonstrates that the injector successfully worked in SFX of the human A2A adenosine receptor complexed with an antagonist, ZM241385, in lipidic cubic phase and for hen egg-white lysozyme microcrystals in a grease carrier. The injection device has also been applied to many kinds of proteins, not only for static structural analyses but also for dynamics studies using pump–probe techniques.

Over the past decade, ptychography has been proven to be a robust tool for non-destructive high-resolution quantitative electron, X-ray and optical microscopy. It allows for quantitative reconstruction of the specimen's transmissivity, as well as recovery of the illuminating wavefront. Additionally, various algorithms have been developed to account for systematic errors and improved convergence. With fast ptychographic microscopes and more advanced algorithms, both the complexity of the reconstruction task and the data volume increase significantly. PtychoShelves is a software package which combines high-level modularity for easy and fast changes to the data-processing pipeline, and high-performance computing on CPUs and GPUs.

New fitting analyses of two-dimensional diffraction-spot shapes are demonstrated to evaluate strain, strain distribution and domain size in a crystalline ultra-thin film. The evaluations are displayed as residual and population maps as a function of strain or domain size.

This paper reports on the two-scale fractal structure of chromatin organization in the nucleus of the HeLa cell.

This work presents the crystal structure determination of two elusive polymorphs of furazidin, an antibacterial agent, employing a combination of crystal structure prediction (CSP) calculations and an NMR crystallography approach. Two previously uncharacterized neat crystal forms, one of which has two symmetry-independent molecules (form I), whereas the other one is a Z' = 1 polymorph (form II), crystallize in P21/c and P1 space groups, respectively, and both are built by different conformers, displaying different intermolecular interactions. It is demonstrated that the usage of either CSP or NMR crystallography alone is insufficient to successfully elucidate the above-mentioned crystal structures, especially in the case of the Z' = 2 polymorph. In addition, cases of serendipitous agreement in terms of 1H or 13C NMR data obtained for the CSP-generated crystal structures different from the ones observed in the laboratory (false-positive matches) are analyzed and described. While for the majority of analyzed crystal structures the obtained agreement with the NMR experiment is indicative of some structural features in common with the experimental structure, the mentioned serendipity observed in exceptional cases points to the necessity of caution when using an NMR crystallography approach in crystal structure determination.

Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by Fc∊RI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and Fc∊RI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with Fc∊RI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.

A recent study of dog bones excavated from archaeological sites on the Channel Islands of California has cast new light on the past ecology of the islands and the impact that domestic dogs--brought to the islands by Native Americans more than 6,000 years ago—may have once had on the islands’ animals and ecosystems.

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New measurements show that the Milky Way is bigger and more massive than previous data suggested, putting us on equal footing with our neighbor. Specifically, the Milky Way is 15 percent larger in size and contains 50 percent more mass. That is the cosmic equivalent of a 5-foot-5, 140-pound man suddenly bulking up to the size of a 6-foot-3, 210-pound NFL linebacker.

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When songwriter Oscar Hammerstein penned the lyrics “fish gotta swim, birds gotta fly,” he clearly did not have the ratites in mind. Large flightless birds, the ratites include ostriches, emus, rheas and cassowaries, along with kiwis and several extinct species.

The post DNA evidence is rearranging the branches of the avian family tree appeared first on Smithsonian Insider.

A new Smithsonian study that examines 10 million years of the evolution of tiny coral-like organisms called cupuladriid bryzoans has revealed that some species of this organism lingered on earth for more than one million years after the event that ultimately caused their extinction: the rising of the Isthmus of Panama.

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Today the Smithsonian Institution announced Eva J. Pell, Senior Vice President for Research and Dean of the Graduate School at Pennsylvania State University, will be the new Under Secretary for Science at the Institution.

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Testing caterpillars’ taste buds is no simple task. Just like your local salad bar, plants in the wild come in different shapes, textures and flavors. Herbivores rely on each of these cues to tell them what to eat. Deciphering this code is Lind’s task. He’s examining 40 different plant species – half invasive, half native.

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A new census of tropical sulfur butterflies (Aphrissa statira) migrating across the Panama Canal has revealed the central role that weather plays in determining why populations of these lemon-yellow insects vary from year to year.

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Nearly 100 years ago, scientists detected the first signs of cosmic rays—subatomic particles (mostly protons) that zip through space at nearly the speed of light. […]

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In a series of ongoing experiments on Barro Colorado Island in the Panama Canal, Kays and other researchers are using camera traps, radio collars and palm nuts with tracking transmitters attached to them to take a closer look at the nut-hoarding strategies of the agouti.

The post Camera traps & radio collars reveal hoarding strategies of the South American agouti appeared first on Smithsonian Insider.

Recently, researchers from Boston University and the Smithsonian Tropical Research Institute in Panama have been taking a closer look at the vibrations that red-eyed treefrog embryos use as cues to trigger early hatching. They discovered that treefrog embryos can evaluate different features of vibrations.

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Chi Cygni pulses once every 408 days. At its smallest diameter of 300 million miles, it becomes mottled with brilliant spots as massive plumes of hot plasma roil its surface. As it expands, Chi Cygni cools and dims, growing to a diameter of 480 million miles—large enough to engulf and cook our solar system out to the asteroid belt.

The post Distant, dying star gives astronomers preview of the fate of our Sun appeared first on Smithsonian Insider.

Birds do it. Bees do it. And in a laboratory in northern California, scientists using bumblebees recently figured out the best way to measure it--vertical lift!

The post Laboratory tests reveal precise way to measure vertical lift in bumblebees and other small insects and birds appeared first on Smithsonian Insider.