Electron Microdiffraction Research Articles

A Fully Saturated Covalent Organic Framework.

We report the design and synthesis of the first aliphatic covalent organic framework (COF), NUS-119, and its subsequent conversion to NUS-120, marking the first fully saturated COF. NUS-119 is built by imine-linkages exhibiting high crystallinity and porosity, achieved by using a Lewis acid as a reaction modulator to circumvent compatibility issues between the Brønsted acid and the strong basic monomer. The structure was successfully solved using 3D microelectron diffraction (microED) techniques. NUS-119 and NUS-120 demonstrated remarkable catalytic performance in base-catalyzed Knoevenagel condensation reactions, exhibiting high conversion rates, excellent size selectivity, and good recyclability. This work advances the understanding of COF materials and paves the way for future research and applications.

Hexagons on rectangles: Epitaxial graphene on Ru[formula omitted]

Ruthenium is emerging as a promising candidate to replace copper in highly integrated electronics by enabling barrierless metallization in ultrathin interconnects. From this perspective, the study of graphene growth on such surface templates is of paramount importance as a platform for graphene integration in electronic devices. In particular, graphene growth on the Ru(101‾0) surface allows selective growth of different graphene orientations, one-dimensional structures, and reduced substrate interaction compared to the well-established hexagonal Ru(0001) substrate. Real-time growth observations using low-energy electron microscopy and micro-diffraction highlight the influence of substrate symmetry on graphene growth, leading to the formation of rectangular islands with distinct zigzag- or armchair-terminated edges. Bilayer formation on Ru(101‾0) occurs by nucleation of graphene nanoribbons under the monolayer. Micro-spot angle-resolved photoemission spectroscopy shows significantly less charge-transfer doping in these freestanding, zigzag-terminated bilayer graphene nanoribbons, indicating reduced graphene-substrate interaction and hence more effective decoupling as compared to graphene/Ru(0001). Our results show that the growth of graphene on non-hexagonal substrates opens new pathways for tailoring the graphene-substrate interaction at the interface, and thus the properties of graphene beyond the limits imposed by hexagonal substrates.

Design and fabrication of 3D-printed in situ crystallization plates for probing microcrystals in an external electric field

X-ray crystallography is an established tool to probe the structure of macromolecules with atomic resolution. Compared with alternative techniques such as single-particle cryo-electron microscopy and micro-electron diffraction, X-ray crystallography is uniquely suited to room-temperature studies and for obtaining a detailed picture of macromolecules subjected to an external electric field (EEF). The impact of an EEF on proteins has been extensively explored through single-crystal X-ray crystallography, which works well with larger high-quality protein crystals. This article introduces a novel design for a 3D-printed in situ crystallization plate that serves a dual purpose: fostering crystal growth and allowing the concurrent examination of the effects of an EEF on crystals of varying sizes. The plate's compatibility with established X-ray crystallography techniques is evaluated.

AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography.

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

Microelectron Diffraction Reveals Contamination of Polymorphs by Structure Analysis of Microscale Crystals in a Novel Pharmaceutical Cocrystal

Microelectron Diffraction Reveals Contamination of Polymorphs by Structure Analysis of Microscale Crystals in a Novel Pharmaceutical Cocrystal

Pharmaceutical Development Challenges in a Beyond Rule of Five Prodrug: Case Study of ABBV-167, Phosphate Prodrug of Venetoclax.

ABBV-167, a phosphate prodrug of BCL-2 inhibitor venetoclax, was recently progressed into the clinic as an alternative means of reducing pill burden for patients in high-dose indications. The dramatically enhanced aqueous solubility of ABBV-167 allowed for high drug loading within a crystalline tablet and, when administered in phase I clinical study, conferred venetoclax exposure commensurate with the equivalent dose administered as an amorphous solid dispersion. In enabling the progression into the clinic, we performed a comprehensive evaluation of the CMC development aspects of this beyond the rule of five (bRo5) prodrug. Adding a phosphate moiety resulted in excessively complex chemical speciation and solid form landscapes with significant physical-chemical stability liabilities. A combination of experimental and computational methods including microelectron diffraction (MicroED), total scattering, tablet colorimetry, finite element, and molecular dynamics modeling were used to understand CMC developability across drug substance and product manufacture and storage. The prodrug's chemical structural characteristics and loose crystal packing were found to be responsible for the loss of crystallinity during its manufacturing, which in turn led to high solid-state chemical reactivity and poor shelf life stability. The ABBV-167 case exemplifies key CMC development challenges for complex chemical matter such as bRo5 phosphate prodrugs with significant ramifications during drug substance and drug product manufacturing and storage.

Micro-electron diffraction applications in the structural analysis of metal–organic frameworks and amorphous phases prepared by mechanochemistry

Micro-electron diffraction applications in the structural analysis of metal–organic frameworks and amorphous phases prepared by mechanochemistry

A complete micro-electron diffraction (MicroED) solution for fast structure determination of macromolecules and small molecules

A complete micro-electron diffraction (MicroED) solution for fast structure determination of macromolecules and small molecules

Structure Elucidation of Olanzapine Molecular Salts by Combining Mechanochemistry and Micro-Electron Diffraction

Structure Elucidation of Olanzapine Molecular Salts by Combining Mechanochemistry and Micro-Electron Diffraction

Caryochroite, a Rare Mineral from the Titanosilicate Group: Crystal Structure, Crystal Chemistry, and Thermal Properties

The crystal structure of the rare mineral caryochroite is determined for the first time using X-ray diffractometry and electron microdiffraction data. The new idealized crystal formula of the mineral is [Na(Sr0.5Ca0.5)Mg]3[Fe\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{8}^{{3 + }}$$\\end{document}Mn(\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ ext{Fe}}_{{0.5}}^{{2 + }}{{\\square }_{{0.5}}}$$\\end{document})]10(Ti2Si12)O37(OH)14(Н2О)3. The parameters of the monoclinic unit cell refined by the least square method are a 16.550(3), b 5.281(2), c 24.25(3) Å, β 93.0°, Z = 2, space group P2/n. The crystal structure of caryochroite is a new type of Ti–Si lattice responsible for the large structural channels, which are able to enclose the exchange cations and a high amount of water components (OH groups and H2O molecules).

Levocetirizin‐Dihydrochlorid: Die Aufklärung einer lang verborgenen Struktur mittels Elektronenbeugung

AbstractLevocetirizin ist ein oral verabreichtes Antihistaminikum der zweiten Generation, dass seit über 25 Jahren zur Behandlung von Allergiesymptomen und langanhaltender Nesselsucht eingesetzt wird. Trotz der weiten Verbreitung dieser Verbindung war ihre Kristallstruktur bisher unbekannt. Hier berichten wir über die Anwendung der 3D‐Elektronenbeugung (3D ED)/Mikrokristallbeugung (MicroED) zur Bestimmung der Kristallstruktur von Levocetirizindihydrochlorid direkt aus kristallinen Pulvern, handelsüblicher Tabletten, die diese Verbindung enthalten. Weiterhin zeigen wir auch den Nutzen der dynamischen Verfeinerung zur eindeutigen Zuordnung der absoluten Konfiguration. Die Ergebnisse unterstreichen das immense Potenzial der 3D‐ED/MicroED für die Strukturaufklärung von Komponenten mikrokristalliner Gemische, wodurch sich die Züchtung großer Einkristalle und der Einsatz ergänzender Analysetechniken erübrigt. Dies wird nicht nur dazu beitragen, dass die Methode für die Identifizierung, sondern auch für die primäre strukturelle Charakterisierung neuer Verbindungen in Zukunft an Bedeutung gewinnt.

Mechanochemical Synthesis of Cocrystal: From Mechanism to Application

Cocrystal engineering is gaining interest across various disciplines since it can effectively tune the properties of solid substances via noncovalent synthesis by introducing new components into the lattice. Mechanochemistry is without a doubt the most valuable tool for the research of cocrystals, which combines the pursuit of efficient and sustainable process pathways with the exploration of supramolecular synthons that cannot be discovered using solution methods. In this review, concerning the significance of the mechanochemical synthesis of cocrystals, we begin by outlining the strategies for mechanochemical preparation of cocrystals. We then elaborate on the theoretical mechanisms of the mechanochemically induced formation of cocrystals and their polymorphs. On this foundation, several cross-fields in which mechanochemistry enhances the application value of cocrystal engineering are shown to overcome existing limitations, which are difficult or impossible to access using conventional solution methods. More importantly, we demonstrate that the introduction of new methods, such as cultivating single crystals from melt microdroplets, and new techniques, such as microelectron diffraction (Micro-ED), has harmoniously united the fields of cocrystal engineering and mechanochemistry. Finally, a brief conclusion and outlook are presented, including current challenges and future opportunities for the cooperation of mechanochemistry and cocrystal engineering.

Unraveling van der Waals epitaxy: A real-time in-situ study of MoSe2 growth on graphene/Ru(0001)

In the present work we investigate the growth of monolayer MoSe2 on selenium-intercalated graphene on Ru(0001), a model layered heterostructure combining a transition metal dichalcogenide with graphene, using low energy electron microscopy and micro-diffraction. Real-time observation of MoSe2 on graphene growth reveals the island nucleation dynamics at the nanoscale. Upon annealing, larger islands are formed by sliding and attachment of multiple nanometer-sized MoSe2 flakes. Local micro-spot angle-resolved photoemission spectroscopy reveals the electronic structure of the heterostructure, indicating that no charge transfer occurs within adjacent layers. The observed behavior is attributed to intercalation of Se at the graphene/Ru(0001) interface. The unperturbed nature of the proposed heterostructure therefore renders it as a model system for investigations of graphene supported TMD nanostructures.

Structure of Y0.75La0.25PO4·0.67H2O rhabdophane nanoparticles synthesized by the hydrothermal microwave method

Nanopowders with a rhabdophane structure of nominal composition Y0.75La0.25PO4·0.67H2O were obtained by hydrothermal-microwave synthesis. The monoclinic structure of rhabdophane was refined by the Rietveld method within the C2 (5) space group. The nanopowders were studied by X-ray diffraction (XRD), transmission electron microscopy (TEM) and electron microdiffraction (selected area electron diffraction, SAED), small angle X-ray scattering (SAXS), thermal analysis, low-temperature nitrogen adsorption-desorption measurements for analysis of surface area and porosity (ASAP) and pycnometric density measurements. These methods showed the formation of rod-like single-crystal rhabdophane nanoparticles with isolated pores inside the nanoparticles. To describe the formation of such nanoparticles, a mechanism of coherent-accommodative intergrowth of nanoscale clusters with rhabdophane structure is proposed.

Determining biomolecular structures near room temperature using X-ray crystallography: concepts, methods and future optimization.

For roughly two decades, cryocrystallography has been the overwhelmingly dominant method for determining high-resolution biomolecular structures. Competition from single-particle cryo-electron microscopy and micro-electron diffraction, increased interest in functionally relevant information that may be missing or corrupted in structures determined at cryogenic temperature, and interest in time-resolved studies of the biomolecular response to chemical and optical stimuli have driven renewed interest in data collection at room temperature and, more generally, at temperatures from the protein-solvent glass transition near 200 K to ∼350 K. Fischer has recently reviewed practical methods for room-temperature data collection and analysis [Fischer (2021), Q. Rev. Biophys. 54, e1]. Here, the key advantages and physical principles of, and methods for, crystallographic data collection at noncryogenic temperatures and some factors relevant to interpreting the resulting data are discussed. For room-temperature data collection to realize its potential within the structural biology toolkit, streamlined and standardized methods for delivering crystals prepared in the home laboratory to the synchrotron and for automated handling and data collection, similar to those for cryocrystallography, should be implemented.

Growing and making nano- and microcrystals.

Thanks to recent technological advances in X-ray and micro-electron diffraction and solid-state NMR, structural information can be obtained by using much smaller crystals. Thus, microcrystals have become a valuable commodity rather than a mere stepping stone toward obtaining macroscopic crystals. Microcrystals are particularly useful for structure determination using serial data collection approaches at synchrotrons and X-ray free-electron lasers. The latter's enormous peak brilliance and short X-ray pulse duration mean that structural information can be obtained before the effects of radiation damage are seen; these properties also facilitate time-resolved crystallography. To establish defined reaction initiation conditions, microcrystals with a desired and narrow size distribution are critical. Here, we describe milling and seeding techniques as well as filtration approaches for the reproducible and size-adjustable preparation of homogeneous nano- and microcrystals. Nanocrystals and crystal seeds can be obtained by milling using zirconium beads and the BeadBug homogenizer; fragmentation of large crystals yields micro- or nanocrystals by flowing crystals through stainless steel filters by using an HPLC pump. The approaches can be scaled to generate micro- to milliliter quantities of microcrystals, starting from macroscopic crystals. The procedure typically takes 3-5 d, including the time required to grow the microcrystals.

RCSB Protein Data bank: Tools for visualizing and understanding biological macromolecules in 3D.

Now in its 52nd year of continuous operations, the Protein Data Bank (PDB) is the premiere open-access global archive housing three-dimensional (3D) biomolecular structure data. It is jointly managed by the Worldwide Protein Data Bank (wwPDB) partnership. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) is funded by the National Science Foundation, National Institutes of Health, and US Department of Energy and serves as the US data center for the wwPDB. RCSB PDB is also responsible for the security of PDB data in its role as wwPDB-designated Archive Keeper. Every year, RCSB PDB serves tens of thousands of depositors of 3D macromolecular structure data (coming from macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro-electron diffraction). The RCSB PDB research-focused web portal (RCSB.org) makes PDB data available at no charge and without usage restrictions to many millions of PDB data consumers around the world. The RCSB PDB training, outreach, and education web portal (PDB101.RCSB.org) serves nearly 700 K educators, students, and members of the public worldwide. This invited Tools Issue contribution describes how RCSB PDB (i) is organized; (ii) works with wwPDB partners to process new depositions; (iii) serves as the wwPDB-designated Archive Keeper; (iv) enables exploration and 3D visualization of PDB data via RCSB.org; and (v) supports training, outreach, and education via PDB101.RCSB.org. New tools and features at RCSB.org are presented using examples drawn from high-resolution structural studies of proteins relevant to treatment of human cancers by targeting immune checkpoints.

Protein Data Bank: A Comprehensive Review of 3D Structure Holdings and Worldwide Utilization by Researchers, Educators, and Students.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), funded by the United States National Science Foundation, National Institutes of Health, and Department of Energy, supports structural biologists and Protein Data Bank (PDB) data users around the world. The RCSB PDB, a founding member of the Worldwide Protein Data Bank (wwPDB) partnership, serves as the US data center for the global PDB archive housing experimentally-determined three-dimensional (3D) structure data for biological macromolecules. As the wwPDB-designated Archive Keeper, RCSB PDB is also responsible for the security of PDB data and weekly update of the archive. RCSB PDB serves tens of thousands of data depositors (using macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro-electron diffraction) annually working on all permanently inhabited continents. RCSB PDB makes PDB data available from its research-focused web portal at no charge and without usage restrictions to many millions of PDB data consumers around the globe. It also provides educators, students, and the general public with an introduction to the PDB and related training materials through its outreach and education-focused web portal. This review article describes growth of the PDB, examines evolution of experimental methods for structure determination viewed through the lens of the PDB archive, and provides a detailed accounting of PDB archival holdings and their utilization by researchers, educators, and students worldwide.

Micro-electron diffraction structure of the aggregation-driving N terminus of Drosophila neuronal protein Orb2A reveals amyloid-like β-sheets

Amyloid protein aggregation is commonly associated with progressive neurodegenerative diseases, however not all amyloid fibrils are pathogenic. The neuronal cytoplasmic polyadenylation element binding protein is a regulator of synaptic mRNA translation and has been shown to form functional amyloid aggregates that stabilize long-term memory. In adult Drosophila neurons, the cytoplasmic polyadenylation element binding homolog Orb2 is expressed as 2 isoforms, of which the Orb2B isoform is far more abundant, but the rarer Orb2A isoform is required to initiate Orb2 aggregation. The N terminus is a distinctive feature of the Orb2A isoform and is critical for its aggregation. Intriguingly, replacement of phenylalanine in the fifth position of Orb2A with tyrosine (F5Y) in Drosophila impairs stabilization of long-term memory. The structure of endogenous Orb2B fibers was recently determined by cryo-EM, but the structure adopted by fibrillar Orb2A is less certain. Here we use micro-electron diffraction to determine the structure of the first 9 N-terminal residues of Orb2A, at a resolution of 1.05 Å. We find that this segment (which we term M9I) forms an amyloid-like array of parallel in-register β-sheets, which interact through side chain interdigitation of aromatic and hydrophobic residues. Our structure provides an explanation for the decreased aggregation observed for the F5Y mutant and offers a hypothesis for how the addition of a single atom (the tyrosyl oxygen) affects long-term memory. We also propose a structural model of Orb2A that integrates our structure of the M9I segment with the published Orb2B cryo-EM structure.

Four-dimensional emittance measurements of ultrafast electron diffraction optics corrected up to sextupole order

Ultrafast electron diffraction (UED) is a technique in which short-pulse electron beams can probe the femtosecond-scale evolution of atomic structure in matter driven far from equilibrium. As an accelerator physics challenge, UED imposes stringent constraints on the brightness of the probe electron beam. The low normalized emittance employed in UED, often at the 10-nm scale and below, is particularly sensitive to both applied field aberrations and space-charge effects. The role of aberrations is increasingly important in small probe systems that often feature multiple orders of magnitude variations in beam size during transport. In this work, we report the correction of normal quadrupole, skew quadrupole, and sextupole aberrations via dedicated corrector elements in an ultrafast electron microdiffraction beamline. To do this, we generate precise four-dimensional phase space maps of rf-compressed electron beams and find excellent agreement with aberration-free space-charge simulations. Finally, we discuss the role a probe-forming aperture can play in improving the brightness of bunches with appreciable space-charge effects.