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Until recently, structure determination by transmission electron microscopy of beam-sensitive three-dimensional nanocrystals required electron diffraction tomography data collection at liquid-nitrogen temperature, in order to reduce radiation damage. Here it is shown that the novel Timepix detector combines a high dynamic range with a very high signal-to-noise ratio and single-electron sensitivity, enabling ab initio phasing of beam-sensitive organic compounds. Low-dose electron diffraction data (∼0.013 e− Å−2 s−1) were collected at room temperature with the rotation method. It was ascertained that the data were of sufficient quality for structure solution using direct methods using software developed for X-ray crystallography ( XDS, SHELX) and for electron crystallography ( ADT3D/ PETS, SIR2014). [ABSTRACT FROM AUTHOR]

C-H functionalization of purely aliphatic substrates is a challenging endeavor, as the absence of directing groups generally thwarts attempts at regiocontrol. This is particularly true for difunctionalization reactions, where the control of relative stereochemistry poses an additional obstacle. The Baddeley reaction of decalins, despite suffering from strong limitations with regard to yield and generality, stands as one of only few known transformations capable of regio- and stereocontrol in aliphatic C-H functionalization. Herein, we report a regio- and diastereoselective method for the double functionalization of decalins enabling access to a novel, unreported regioisomer in synthetically useful yields. This method was also successfully applied to a range of other alkane substrates, enabling a straightforward synthesis of keto alcohols from the simplest alkane building blocks., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Exciting developments are unfolding in the realm of chemical crystallography, especially with the profound impact of electron diffraction and the remarkable progress it has witnessed in recent years.

Cobalt complexes with multiproton- and multielectron-responsive ligands are of interest for challenging catalytic transformations. The chemical and redox noninnocence of pentane-2,4-dione bis( S -methylisothiosemicarbazone) (PBIT) in a series of cobalt complexes has been studied by a range of methods, including spectroscopy [UV-vis, NMR, electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS)], cyclic voltammetry, X-ray diffraction, and density functional theory (DFT) calculations. Two complexes [Co III (H 2 L SMe )I]I and [Co III ( L SMe )I 2 ] were found to act as precatalysts in a Wacker-type oxidation of olefins using phenylsilane, the role of which was elucidated through isotopic labeling. Insights into the mechanism of the catalytic transformation as well as the substrate scope of this selective reaction are described, and the essential role of phenylsilane and the noninnocence of PBIT are disclosed. Among the several relevant species characterized was an unprecedented Co(III) complex with a dianionic diradical PBIT ligand ([Co III (L SMe•• )I])., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Unveiling the coke formation in zeolites is an essential prerequisite for tackling the deactivation of these catalysts in the transformations of hydrocarbons. Herein, we present the direct mapping of coke in the micropores of ZSM-5 catalysts used in methanol-to-hydrocarbons conversion by single-crystal electron diffraction analysis. The latter technique revealed a polycyclic aromatic structure along the straight channel, wherein the high-quality data permit refinement of its occupancy to about 40 %. These findings were exploited to analyze the evolution of micropore coke during the reaction. Herein, coke-associated signals, which correlate with the activity loss, indicate that the nucleation of coke commences in the intersections of sinusoidal and straight channels, while the formation of coke in the straight pores occurs in the late stages of deactivation. The findings uncover an attractive method for analyzing coke deposition in the micropore domain., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Electron diffraction enables structure determination of organic small molecules using crystals that are too small for conventional X-ray crystallography. However, because of uncertainties in the experimental parameters, notably the detector distance, the unit-cell parameters and the geometry of the structural models are typically less accurate and precise compared with results obtained by X-ray diffraction. Here, an iterative procedure to optimize the unit-cell parameters obtained from electron diffraction using idealized restraints is proposed. The cell optimization routine has been implemented as part of the structure refinement, and a gradual improvement in lattice parameters and data quality is demonstrated. It is shown that cell optimization, optionally combined with geometrical corrections for any apparent detector distortions, benefits refinement of electron diffraction data in small-molecule crystallography and leads to more accurate structural models., (© Tim Gruene et al. 2022.)

In the past few years, many exciting papers reported results based on crystal structure determination by electron diffraction. The aim of this review is to provide general and practical information to structural chemists interested in stepping into this emerging field. We discuss technical characteristics of electron microscopes for research units that would like to acquire their own instrumentation, as well as those practical aspects that appear different between X-ray and electron crystallography. We also include a discussion about applications where electron crystallography provides information that is different, and possibly complementary, with respect to what is available from X-ray crystallography.

The emerging field of 3D electron diffraction (3D ED) opens new opportunities for structure determination from sub-micrometre-sized crystals. Although the foundations of this technology emerged earlier, the past decade has seen developments in cryo-electron microscopy and (X-ray) crystallography that particularly enable the widespread use of 3D ED. This Perspective describes to chemists and chemical crystallographers just how similar electron and X-ray diffraction are and discusses their complementary aspects. We wish to establish 3D ED in the broader chemistry community, such that electron crystallography becomes a common part of the analytical chemistry toolkit. With a suitable instrument at their disposal, every skilled crystallographer can quickly learn to perform structure determinations using 3D ED., (© 2021. Springer Nature Limited.)

The costimulatory molecule CD80 is an early marker for immune activation. It is upregulated on activated antigen-presenting cells. We aimed at developing a tracer for imaging CD80 by positron emission tomography (PET). Novel CD80 ligands were synthesized and tested by SPR for affinity to human CD80 (hCD80) and displacement of endogenous ligands. Several compounds bound with one-digit nanomolar affinity to hCD80 and displaced CTLA-4 and CD28 at nanomolar concentrations. A structure-affinity relationship study revealed relevant moieties for strong affinity to hCD80 and positions for further modifications. Lead compound MT107 ( 7f ) was radiolabeled with carbon-11. In vitro, [ 11 C]MT107 showed specific binding to hCD80-positive tissue and high plasma protein binding. In vivo, [ 11 C]MT107 accumulated in liver, gall bladder, and intestines but only scarcely in hCD80-positive xenografts. The unfavorable in vivo performance may result from high plasma protein binding and extensive biliary excretion.

3D electron crystallography has recently attracted much attention due to its complementarity to X-ray crystallography in determining the structure of compounds from submicrometre sized crystals. A big obstacle lies in obtaining complete data, required for accurate structure determination. Many crystals have a preferred orientation on conventional, flat sample supports. This systematically shades some part of the sample and prevents the collection of complete data, even when several data sets are combined. We introduce two types of three-dimensional sample supports that enable the collection of complete data sets. In the first approach the carbon layer forms coils on the sample support. The second approach is based on chaotic nylon fibres. Both types of grids disrupt the preferred orientation as we demonstrate with a well suited crystal type of MFI-type zeolites. The easy-to-obtain three-dimensional sample supports have different features, ensuring a broad spectrum of applications for these 3D support grids.

3D electron diffraction has reached a stage where the structures of chemical compounds can be solved productively. Instrumentation is lagging behind this development, and to date dedicated electron diffractometers for data collection based on the rotation method do not exist. Current studies use transmission electron microscopes as a workaround. These are optimized for imaging, which is not optimal for diffraction studies. The beam intensity is very high, it is difficult to create parallel beam illumination and the detectors used for imaging are of only limited use for diffraction studies. In this work, the combination of an EIGER hybrid pixel detector with a transmission electron microscope to construct a productive electron diffractometer is described. The construction not only refers to the combination of hardware but also to the calibration of the system, so that it provides rapid access to the experimental parameters that are necessary for processing diffraction data. Until fully integrated electron diffractometers become available, this describes a setup for productive and efficient operation in chemical crystallography., (open access.)

Compared with X-rays, electron diffraction faces a crucial challenge: dynamical electron scattering compromises structure solution and its effects can only be modelled in specific cases. Dynamical scattering can be reduced experimentally by decreasing crystal size but not without a penalty, as it also reduces the overall diffracted intensity. In this article it is shown that nanometre-sized crystals from organic pharmaceuticals allow positional refinement of the hydrogen atoms, even whilst ignoring the effects of dynamical scattering during refinement. To boost the very weak diffraction data, a highly sensitive hybrid pixel detector was employed. A general likelihood-based computational approach was also introduced for further reducing the adverse effects of dynamic scattering, which significantly improved model accuracy, even for protein crystal data at substantially lower resolution., (open access.)

Chemists of all fields currently publish about 50 000 crystal structures per year, the vast majority of which are X-ray structures. We determined two molecular structures by employing electron rather than X-ray diffraction. For this purpose, an EIGER hybrid pixel detector was fitted to a transmission electron microscope, yielding an electron diffractometer. The structure of a new methylene blue derivative was determined at 0.9 Å resolution from a crystal smaller than 1×2 μm 2 . Several thousand active pharmaceutical ingredients (APIs) are only available as submicrocrystalline powders. To illustrate the potential of electron crystallography for the pharmaceutical industry, we also determined the structure of an API from its pill. We demonstrate that electron crystallography complements X-ray crystallography and is the technique of choice for all unsolved cases in which submicrometer-sized crystals were the limiting factor., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Corrections are made to Table 1 in the article by van Genderen et al. [Acta Cryst. (2016), A72, 236-242]., (open access.)

Electron diffraction is a relatively novel alternative to X-ray crystallography for the structure determination of macromolecules from three-dimensional nanometre-sized crystals. The continuous-rotation method of data collection has been adapted for the electron microscope. However, there are important differences in geometry that must be considered for successful data integration. The wavelength of electrons in a TEM is typically around 40 times shorter than that of X-rays, implying a nearly flat Ewald sphere, and consequently low diffraction angles and a high effective sample-to-detector distance. Nevertheless, the DIALS software package can, with specific adaptations, successfully process continuous-rotation electron diffraction data. Pathologies encountered specifically in electron diffraction make data integration more challenging. Errors can arise from instrumentation, such as beam drift or distorted diffraction patterns from lens imperfections. The diffraction geometry brings additional challenges such as strong correlation between lattice parameters and detector distance. These issues are compounded if calibration is incomplete, leading to uncertainty in experimental geometry, such as the effective detector distance and the rotation rate or direction. Dynamic scattering, absorption, radiation damage and incomplete wedges of data are additional factors that complicate data processing. Here, recent features of DIALS as adapted to electron diffraction processing are shown, including diagnostics for problematic diffraction geometry refinement, refinement of a smoothly varying beam model and corrections for distorted diffraction images. These novel features, combined with the existing tools in DIALS, make data integration and refinement feasible for electron crystallography, even in difficult cases., (open access.)

Electron-diffraction data on the zeolites Silicalite-1 and ZSM-5 (both MFI framework type) were collected from individual grains of about 150×100×50 nm 3 . Crystals were synthesized with tetrapropylammonium as structure-directing agent. The resolution extended to about 0.8 Å for Silicalite-1 and about 0.9-1.0 Å for ZSM-5 crystals. Analysis of several data sets showed that at the nanometre-scale, these zeolite crystals are single crystals and not intergrown., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electron crystallography is a discipline that currently attracts much attention as method for inorganic, organic and macromolecular structure solution. EIGER, a direct-detection hybrid pixel detector developed at the Paul Scherrer Institut, Switzerland, has been tested for electron diffraction in a transmission electron microscope. EIGER features a pixel pitch of 75 × 75 µm 2 , frame rates up to 23 kHz and a dead time between frames as low as 3 µs. Cluster size and modulation transfer functions of the detector at 100, 200 and 300 keV electron energies are reported and the data quality is demonstrated by structure determination of a SAPO-34 zeotype from electron diffraction data.

The brain is highly plastic and undergoes changes in response to many experiences. Learning especially can induce structural remodeling of dendritic spines, which is thought to relate to memory formation. Classical Pavlovian fear conditioning (FC) traditionally pairs an auditory cue with an aversive footshock, and has been widely used to study neural processes underlying associative learning and memory. Past research has found dendritic spine changes after FC in several structures. But, due to heterogeneity of cells within brain structures and limitations of traditional neuroanatomical techniques, it is unclear if all cells included in analyses were actually active during learning processes, even if known circuits are isolated. In this study, we employed a novel approach to analyze structural plasticity explicitly in neurons activated by exposure to either cued or uncued footshocks. We used male and female Arc-dVenus transgenic mice, which express the Venus fluorophore driven by the activity-related Arc promoter, to identify neurons that were active during either scenario. We then targeted fluorescent microinjections to Arc+ and neighboring Arc- neurons in the basolateral area of the amygdala (BLA) and auditory association cortex (TeA). In both BLA and TeA, Arc+ neurons had reduced thin and mushroom spine densities compared to Arc- neurons. This effect was present in males and females alike and also in both cued and uncued shock groups. Overall, this study adds to our understanding of how neuronal activity affects structural plasticity, and represents a methodological advance in the ways we can directly relate structural changes to experience-related neural activity., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Until recently, structure determination by transmission electron microscopy of beam-sensitive three-dimensional nanocrystals required electron diffraction tomography data collection at liquid-nitrogen temperature, in order to reduce radiation damage. Here it is shown that the novel Timepix detector combines a high dynamic range with a very high signal-to-noise ratio and single-electron sensitivity, enabling ab initio phasing of beam-sensitive organic compounds. Low-dose electron diffraction data (∼ 0.013 e(-) Å(-2) s(-1)) were collected at room temperature with the rotation method. It was ascertained that the data were of sufficient quality for structure solution using direct methods using software developed for X-ray crystallography (XDS, SHELX) and for electron crystallography (ADT3D/PETS, SIR2014).

The crystallographic reliability index [Formula: see text] is based on a method proposed more than two decades ago. Because its calculation is computationally expensive its use did not spread into the crystallographic community in favor of the cross-validation method known as [Formula: see text]. The importance of [Formula: see text] has grown beyond a pure validation tool. However, its application requires a sufficiently large dataset. In this work we assess the reliability of [Formula: see text] and we compare it with k-fold cross-validation, bootstrapping, and jackknifing. As opposed to proper cross-validation as realized with [Formula: see text], [Formula: see text] relies on a method of reducing bias from the structural model. We compare two different methods reducing model bias and question the widely spread notion that random parameter shifts are required for this purpose. We show that [Formula: see text] has as little statistical bias as [Formula: see text] with the benefit of a much smaller variance. Because the calculation of [Formula: see text] is based on the entire dataset instead of a small subset, it allows the estimation of maximum likelihood parameters even for small datasets. [Formula: see text] enables maximum likelihood-based refinement to be extended to virtually all areas of crystallographic structure determination including high-pressure studies, neutron diffraction studies, and datasets from free electron lasers.

Potassium channels selectively conduct K(+) ions across cellular membranes with extraordinary efficiency. Their selectivity filter exhibits four binding sites with approximately equal electron density in crystal structures with high K(+) concentrations, previously thought to reflect a superposition of alternating ion- and water-occupied states. Consequently, cotranslocation of ions with water has become a widely accepted ion conduction mechanism for potassium channels. By analyzing more than 1300 permeation events from molecular dynamics simulations at physiological voltages, we observed instead that permeation occurs via ion-ion contacts between neighboring K(+) ions. Coulomb repulsion between adjacent ions is found to be the key to high-efficiency K(+) conduction. Crystallographic data are consistent with directly neighboring K(+) ions in the selectivity filter, and our model offers an intuitive explanation for the high throughput rates of K(+) channels., (Copyright © 2014, American Association for the Advancement of Science.)

Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

The ability of many copper metalloenzymes to activate O2 and transfer it to organic substrates has motivated extensive attention in the literature. Investigations focusing on synthetic analogues have provided a detailed understanding of the structures of potential intermediates, thereby helping to guide mechanistic studies. We report herein a crystallographically characterized synthetic Cu(II)2(μ-η(1):η(1)-O2) complex exhibiting cis-peroxo bonding geometry, known in iron chemistry but previously unobserved for copper. Detailed investigation by UV-vis, resonance Raman, and infrared spectroscopies provides evidence for a significantly diminished copper-oxygen interaction (ε ≈ 3000 M(-1) cm(-1), ν(Cu-O) = 437 cm(-1), ν(O-O) = 799 cm(-1)) relative to those in known 'coupled' Cu2O2 species, consistent with magnetic measurements which show that the peroxide mediates only weak antiferromagnetic coupling (-2J = 144 cm(-1)). These characteristics are comparable with those of a computationally predicted transition state for O2 binding to type 3 copper centers, providing experimental evidence for the proposed mechanism of O2 activation and supporting the biological relevance of the Cu(II)2(μ-η(1):η(1)-O2) cis-species. The peroxide bonding arrangement also allows binding of sodium cations, observed both in the solid state and in solution. Binding induces changes on an electronic level, as monitored by UV-vis spectroscopy (K(a) = 1700 M(-1)), reminiscent of redox-inactive metal binding by iron-oxygen species. The results presented highlight the analogous chemistry these reactive oxygen species undergo, with respect to both their mechanism of formation, and the molecular interactions in which they participate.

Some of the improvements in SHELX2013 make SHELXL convenient to use for refinement of macromolecular structures against neutron data without the support of X-ray data. The new NEUT instruction adjusts the behaviour of the SFAC instruction as well as the default bond lengths of the AFIX instructions. This work presents a protocol on how to use SHELXL for refinement of protein structures against neutron data. It includes restraints extending the Engh & Huber [ Acta Cryst. (1991), A 47 , 392-400] restraints to H atoms and discusses several of the features of SHELXL that make the program particularly useful for the investigation of H atoms with neutron diffraction. SHELXL2013 is already adequate for the refinement of small molecules against neutron data, but there is still room for improvement, like the introduction of chain IDs for the refinement of macromolecular structures.

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we present the crystal structure of Pol I from Saccharomyces cerevisiae at 3.0 Å resolution. The Pol I structure shows a compact core with a wide DNA-binding cleft and a tightly anchored stalk. An extended loop mimics the DNA backbone in the cleft and may be involved in regulating Pol I transcription. Subunit A12.2 extends from the A190 jaw to the active site and inserts a transcription elongation factor TFIIS-like zinc ribbon into the nucleotide triphosphate entry pore, providing insight into the role of A12.2 in RNA cleavage and Pol I insensitivity to α-amanitin. The A49-A34.5 heterodimer embraces subunit A135 through extended arms, thereby contacting and potentially regulating subunit A12.2.

Model building starting from, for example, a molecular-replacement solution with low sequence similarity introduces model bias, which can be difficult to detect, especially at low resolution. The program mrtailor removes low-similarity regions from a template PDB file according to sequence similarity between the target sequence and the template sequence and maps the target sequence onto the PDB file. The modified PDB file can be used to generate external restraints for low-resolution refinement with reduced model bias and can be used as a starting point for model building and refinement. The program can call ProSMART [Nicholls et al. (2012), Acta Cryst. D68, 404-417] directly in order to create external restraints suitable for REFMAC5 [Murshudov et al. (2011), Acta Cryst. D67, 355-367]. Both a command-line version and a GUI exist.

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein-protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.

Medium- to high-resolution X-ray structures of DNA and RNA molecules were investigated to find geometric properties useful for automated model building in crystallographic electron-density maps. We describe a simple method, starting from a list of electron-density 'blobs', for identifying backbone phosphates and nucleic acid bases based on properties of the local electron-density distribution. This knowledge should be useful for the automated building of nucleic acid models into electron-density maps. We show that the distances and angles involving C1' and the P atoms, using the pseudo-torsion angles \eta' and \theta\,' that describe the ...P-C1'-P-C1'... chain, provide a promising basis for building the nucleic acid polymer. These quantities show reasonably narrow distributions with asymmetry that should allow the direction of the phosphate backbone to be established.