3D Electron Diffraction for Chemical Analysis: Instrumentation Developments and Innovative Applications.

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.

Electron Diffraction Enables the Mapping of Coke in ZSM-5 Micropores Formed during Methanol-to-Hydrocarbons Conversion.

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.

Crystal structure of the 14-subunit RNA polymerase I.

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.

Characterization at the Level of Individual Crystals: Single-Crystal MFI Type Zeolite Grains.

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 nm3 . Crystals were synthesized with tetrapropylammonium as structure-directing agent. The resolution extended to about 0.8 Šfor Silicalite-1 and about 0.9-1.0 Šfor ZSM-5 crystals. Analysis of several data sets showed that at the nanometre-scale, these zeolite crystals are single crystals and not intergrown.

New method to compute Rcomplete enables maximum likelihood refinement for small datasets.

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.

Rapid Structure Determination of Microcrystalline Molecular Compounds Using Electron Diffraction.

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 Šresolution from a crystal smaller than 1×2 µm2 . 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.

Solving the RNA polymerase I structural puzzle.

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 Šresolution, 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.

Weakly coupled biologically relevant Cu(II)2(µ-η¹:η¹-O2) cis-peroxo adduct that binds side-on to additional metal ions.

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.

Data collection is your last experiment.

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.

Chemical and Redox Noninnocence of Pentane-2,4-dione Bis(S-methylisothiosemicarbazone) in Cobalt Complexes and Their Application in Wacker-Type Oxidation.

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 [CoIII(H2LSMe)I]I and [CoIII(LSMe)I2] 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 ([CoIII(LSMe••)I]).

mrtailor: a tool for PDB-file preparation for the generation of external restraints.

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.

CELLOPT: improved unit-cell parameters for electron diffraction data of small-molecule crystals.

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.

Integrated analysis of the conformation of a protein-linked spin label by crystallography, EPR and NMR spectroscopy.

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.

Establishing electron diffraction in chemical 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.

The magic triangle goes MAD: experimental phasing with a bromine derivative.

Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine-carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

Reducing dynamical electron scattering reveals hydrogen atoms.

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.

3D-structured supports create complete data sets for electron crystallography.

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.

Synthesis and Structure-Affinity Relationship of Small Molecules for Imaging Human CD80 by Positron Emission Tomography.

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, [11C]MT107 showed specific binding to hCD80-positive tissue and high plasma protein binding. In vivo, [11C]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.

Design guidelines for an electron diffractometer for structural chemistry and structural biology.

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.

A magic triangle for experimental phasing of macromolecules.

Obtaining phase information for the solution of macromolecular structures is still one of the bottlenecks in X-ray crystallography. 5-Amino-2,4,6-triiodoisophthalic acid (I3C), in which three covalently bound iodines form an equilateral triangle, was incorporated into proteins in order to obtain phases by single-wavelength anomalous dispersion (SAD). An improved binding capability compared with simple heavy-metal ions, ready availability, improved recognition of potential heavy-atom sites and low toxicity make I3C particularly suitable for experimental phasing.