Since its launch in 2021, the XtaLAB Synergy-ED has produced many structures, with over 500 unique structures from Rigaku labs alone. CrysAlisPro implements all essential workflows for screening, data collection and data reduction. Recent developments focused on making electron diffraction as easy as X-ray diffraction, allowing novice users to transition from X-ray workflows swiftly. The update will focus on the crystallographic user perspective in operating our JEOL/Rigaku electron diffractometer: The advanced GUI simplifies the interaction with the electron optics for detector distance, beam strength, ultra-low dose operation (flash mode) and easy transition between visual and diffraction modes.The data collection separates screening and complete collection operations with tuneable concurrent analysis.The automation by queue operation now offers automatic z-centring to further reduce the interactive operation of the instrument.Data reduction now includes expanded export for dynamic refinement with Jana2020 and Olex2 AutoChem.Merging tools are improved and can be used with external cluster analysis tools.The results viewer is expanded to handle multi-grain workflows.Simple instrument calibration. Furthermore, the essentials of operating AutoChem Olex2 with kinematical and dynamical data will be highlighted.

Serial electron diffraction (SerialED), which applies a snapshot data acquisition strategy on each crystal, was introduced to tackle the problem of radiation damage in the structure determination of beam-sensitive materials by three-dimensional electron diffraction (3D ED). The snapshot data acquisition in SerialED can be realized both in transmission and scanning transmission electron microscopes (TEM/STEM). However, the current SerialED workflow based on STEM setups requires special external devices and software, which brings challenges for its broader adoption. Here, we present a simplified experimental implementation of STEM-based SerialED on Thermo Fisher Scientific STEMs using common proprietary software interfaced through Python scripts to automate data collection. Specifically, we utilize TEM Imaging and Analysis (TIA) scripting and TEM scripting to access the STEM functionalities of the microscope, and DigitalMicrograph (DM) scripting to control the camera for snapshot data acquisition. Data analysis adapts the existing workflow using the software CrystFEL developed for serial X-ray crystallography. Our workflow for SerialED can be used on any Gatan or Thermo Fisher Scientific camera. We apply this workflow to collect high-resolution STEM SerialED data from two aluminosilicate zeolites, Zeolite Y and ZSM-25, and demonstrate, for the first time, ab initio structure determination through direct methods using the STEM SerialED data. Zeolite Y is relatively stable under the electron beam, and SerialED data extend to 0.60 Å. We show that the structural model obtained using SerialED data merged from 358 crystals is nearly identical to that using continuous rotation electron diffraction (cRED) data from one crystal. This demonstrates that accurate structures can be obtained from SerialED. Zeolite ZSM-25 is very beam-sensitive and has a complex structure. We show that SerialED greatly improves data resolution of ZSM-25, compared to serial rotation electron diffraction (SerialRED), from 1.50 Å to 0.90 Å. This allows for the first time the use of standard phasing methods such as direct methods for ab initio structure determination of ZSM-25.

Along with the adoption of three-dimensional electron diffraction (3D ED/MicroED) as a mainstream tool for structure determination from sub-micron single crystals, questions about best practices regarding each step along the workflow, from data collection to structure solutions, arise. In this paper, we discuss three particular aspects of a 3D ED/MicroED experiment which, after hundreds of structures solved in Rigaku’s laboratories, we have found to be important to consider carefully. First, for a representative model system of a hydrated compound (trehalose dihydrate), we show that cryo-transfer of the sample into the diffractometer is an effective means to prevent dehydration, while cooling of the sample without cryo-transfer yields a marginal improvement only. Next, we demonstrate for a small (tyrosine) and a large (clarithromycin) organic compound, how a simplified and fast workflow for dynamical diffraction calculations can determine absolute crystal structures with high confidence. Finally, we discuss considerations and trade-offs for choosing an optimal effective crystal-to-detector distance; while a long distance is mandatory for a protein (thaumatin) example, even a small molecule with difficult diffraction behavior (cystine) yields superior results at longer distances than the one used by default.

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.

Nanozymes are nanomaterials with biocatalytic properties under physiological conditions and are one class of artificial enzymes to overcome the high cost and low stability of natural enzymes. However, surface ligands on nanomaterials will decrease the catalytic activity of the nanozymes by blocking the active sites. To address this limitation, ligand-free PtAg nanoclusters (NCs) are synthesized and applied as nanozymes for various enzyme-mimicking reactions. By taking advantage of the mutual interaction of zeolitic imidazolate frameworks (ZIF-8) and Pt precursors, a good dispersion of PtAg bimetal NCs with a diameter of 1.78 ± 0.1 nm is achieved with ZIF-8 as a template. The incorporation of PtAgNCs in the voids of ZIF-8 is confirmed with structural analysis using the atomic pair-distribution function and powder X-ray diffraction. Importantly, the PtAgNCs present good catalytic activity for various enzyme-mimicking reactions, including peroxidase-/catalase- and oxidase-like reactions. Further, this work compares the catalytic activity between PtAg NCs and PtAg nanoparticles with different compositions and finds that these two nanozymes present a converse dependency of Ag-loading on their activity. This study contributes to the field of nanozymes and presents a potential option to prepare ligand-free bimetal biocatalysts with sizes in the nanocluster regime.

Levocetirizine is an orally administrated, second-generation antihistaminic active pharmaceutical ingredient that has been used to treat symptoms of allergy and long-term hives for over 25 years. Despite the wide use of this compound, its crystal structure has remained unknown. Here we report the application of 3D electron diffraction (3D ED)/Micro-crystal electron diffraction (MicroED) to determine the crystal structure of Levocetirizine dihydrochloride directly from crystalline powders that were extracted from commercially available tablets containing the compound. We also showcase the utility of dynamical refinement to unambiguously assign absolute configuration. The results highlight the immense potential of 3D ED/MicroED for structure elucidation of components of microcrystalline mixtures that obviates the need to grow large-size single crystals and the use of complementary analytical techniques, which could be important for identification as well as for primary structural characterization.

The effect of window material on electron beam induced phenomena in liquid phase electron microscopy (LPEM) is an interesting yet under-explored subject. We have studied the differences of electron beam induced gold nanoparticle (AuNP) growth subject to three encapsulation materials: Silicon Nitride (Si3N4), carbon and formvar. We find Si3N4 liquid cells (LCs) to result in significantly higher AuNP growth yield as compared to LCs employing the other two materials. In all cases, an electrical bias of the entire LC structures significantly affected particle growth. We demonstrate an inverse correlation of the AuNP growth rate with secondary electron (SE) emission from the windows. We attribute these differences at least in part to variations in SE emission dynamics, which is seen as a combination of material and bias dependent SE escape flux (SEEF) and SE return flux (SERF). Furthermore, our model predictions qualitatively match electrochemistry expectations.

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs). Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-β fibrils of mated β-sheets at atomic resolution. Uperin 3.5 formed a 3-blade symmetrical propeller of nine peptides per fibril layer including tight β-sheet interfaces. This cross-β cryo-EM structure complements the cross-α fibril conformation previously determined by crystallography, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked β-sheets allowing a rounded compactness of the fibril. The kinked β-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked Segments) found in human functional amyloids.

Liquid-phase transmission electron microscopy is a technique for simultaneous imaging of the structure and dynamics of specimens in a liquid environment. The conventional sample geometry consists of a liquid layer tightly sandwiched between two Si3N4 windows with a nominal spacing on the order of 0.5 μm. We describe a variation of the conventional approach, wherein the Si3N4 windows are separated by a 10-μm-thick spacer, thus providing room for gas flow inside the liquid specimen enclosure. Adjusting the pressure and flow speed of humid air inside this environmental liquid cell (ELC) creates a stable liquid layer of controllable thickness on the bottom window, thus facilitating high-resolution observations of low mass-thickness contrast objects at low electron doses. We demonstrate controllable liquid thicknesses in the range 160 ± 34 to 340 ± 71 nm resulting in corresponding edge resolutions of 0.8 ± 0.06 to 1.7 ± 0.8 nm as measured for immersed gold nanoparticles. Liquid layer thickness 40 ± 8 nm allowed imaging of low-contrast polystyrene particles. Hydration effects in the ELC have been studied using poly-N-isopropylacrylamide nanogels with a silica core. Therefore, ELC can be a suitable tool for in situ investigations of liquid specimens.

Serial electron diffraction (SerialED) is an emerging technique, which applies the snapshot data-collection mode of serial X-ray crystallography to three-dimensional electron diffraction (3D Electron Diffraction), forgoing the conventional rotation method. Similarly to serial X-ray crystallography, this approach leads to almost complete absence of radiation damage effects even for the most sensitive samples, and allows for a high level of automation. However, SerialED also necessitates new techniques of data processing, which combine existing pipelines for rotation electron diffraction and serial X-ray crystallography with some more particular solutions for challenges arising in SerialED specifically. Here, we introduce our analysis pipeline for SerialED data, and its implementation using the CrystFEL and diffractem program packages. Detailed examples are provided in extensive supplementary code.

Serial electron diffraction (SerialED) (Smeets, Zou and Wan, 2018; Bücker et al. , 2020) is an emerging three-dimensional electron diffraction (3D-ED) method (Gemmi et al. , 2019; Nannenga and Gonen, 2019), where data from a large ensemble of macromolecular or small-molecule nano-crystals is merged into a high-resolution structure solution. Like in serial X-ray crystallography at synchrotrons and X-ray free-electron lasers (Mehrabi et al. , 2020), SerialED is typically performed in the limit of rapidly acquired single or dose-fractionated diffraction shots per diffracting volume, minimizing deleterious effects of radiation damage. However, the flexibility of a scanning TEM (S/TEM) enables a much broader range of data collection strategies, blurring the lines between serial crystallography (SX), scanning electron nano-diffraction (SEND), and massively automated rotation electron diffraction (RED). Furthermore, the flexibility of SerialED along with the large volume and unique nature of the obtained diffraction data, that is, purely two-dimensional reciprocal-space slices of unknown orientation, call for new approaches for data processing (Jiang et al. , 2009; Smeets and Wan, 2017; Bücker, Hogan-Lamarre and Miller,

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

Work in electron-based macromolecular structure determination has grown in importance with the recent developments in rotation electron crystallography [1].It was recently shown that serial electron crystallography (serialED) can be implemented within an electron microscope (TEM) to recover structures from nanocrystalline inorganic molecules [2].This raises questions on the applicability of serialED to organic macromolecules.Such materials present challenges, notably in terms of dose sensitivity and beam quality.Moreover, TEMs have their own limitations: spatial constraints forbid large-travel high-precision stages or equipment for dynamics triggering.We present the design of a dedicated electron beamline for serialED in development.A Schottky field emitter filtered by a 50-m aperture creates a highly-coherent beam.Sub-s pulses are generated through pulsing of the extraction potential and beam blanking.Properties of the beamline are explored through particle-tracking simulations based on realistic representations of the optics from finite-element methods.Macromolecular structures determination in a high-current regime is discussed: considering a fluence threshold of 5 e-/Å 2 , simulations show that a repetition rate of the order of 100 Hz is achievable.Data processing is explored in a proof-of-principle experiment within a TEM.Using softwares developed for x-ray diffraction, such as CrystFEL 4 and CCP4, the structures of hen-egg lysozyme, and granulovirus are recovered at a resolution of 2 Å and 1.8 Å, respectively.

Journal Article Electron Ptychography of Single Biological Macromolecules Get access Philipp M Pelz, Philipp M Pelz Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, GermanyMaterials Science and Engineering, UC Berkeley, Berkeley, CAMolecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, USA Corresponding author: philipp.pelz@berkeley.edu Search for other works by this author on: Oxford Academic Google Scholar Robert Bücker, Robert Bücker Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany Search for other works by this author on: Oxford Academic Google Scholar Georg Ramm, Georg Ramm Monash Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Melbourne, VIC, 3800, AustraliaDepartment of Biochemistry and Molecular Biology, Monash University, Melbourne, AustraliaBiomedicine Discovery Institute, Monash University, Melbourne, Australia Search for other works by this author on: Oxford Academic Google Scholar Hariprasad Venugopal, Hariprasad Venugopal Monash Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Melbourne, VIC, 3800, Australia Search for other works by this author on: Oxford Academic Google Scholar Günther Kassier, Günther Kassier Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany Search for other works by this author on: Oxford Academic Google Scholar Dennis Eggert, Dennis Eggert Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany Search for other works by this author on: Oxford Academic Google Scholar Peng-Han Lu, Peng-Han Lu Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich, Jülich, Germany Search for other works by this author on: Oxford Academic Google Scholar Rafal E Dunin-Borkowski, Rafal E Dunin-Borkowski Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich, Jülich, Germany Search for other works by this author on: Oxford Academic Google Scholar R J Dwayne Miller R J Dwayne Miller Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, GermanyDepartment of Chemistry and Physics, University of Toronto, Toronto, Canada Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 25, Issue S2, 1 August 2019, Pages 72–73, https://doi.org/10.1017/S1431927619001090 Published: 01 August 2019

Serial crystallography, where diffraction snapshots of a large ensemble of randomly oriented crystals are taken, evades the cumulative damage inherent to rotation diffraction techniques.This approach has facilitated the use of sub-micron crystals in latest-generation X-ray sources, making large classes of small, radiation-sensitive systems such as recalcitrant protein crystals or nano-porous materials amenable to crystallographic structure solution [1].On the other hand, electron radiation provides the advantage of a more favorable ratio of elastic scattering to damaging energy deposition by three orders of magnitude over X-rays, enabling another viable path to study such small, sensitive crystals, using transmission electron microscopes (TEMs) [2].We present a new scheme for high-speed, low-dose protein nano-crystallography in a scanning TEM (S/TEM), which combines the benefits of both serial and electron approaches in order to achieve ultimate dose efficiency, while providing a high level of automation and ease of operation.Combining automated real-space mapping of protein crystal positions and morphologies with hardware-synchronized beam positioning and diffraction detection using a hybrid-pixel camera, diffraction snapshots from hundreds of crystals can be acquired per second, at a hit rate exceeding 60%.Results on lysozyme and granulin samples are presented, both of which were solved at a resolution better than 2Å using mostly standard X-ray software, along with a thorough discussion of the data acquisition and analysis pipeline.

Abstract We describe a new method for serial electron diffraction of protein nano-crystals using a conventional S/TEM microscope. Here, randomly dispersed crystals are mapped, and dose-efficient diffraction patterns measured at each identified position for structure determination. This fully automated workflow is suitable for high-throughput applications with acquisition rates of up to 1 kHz and a hit fraction approaching 100 percent. We demonstrate this method by solving the structure of lysozyme and crystalline granulovirus occlusion bodies to a resolution of 1.80 A and 1.55 A, respectively. This method promises to provide rapid high-quality structure determination for all classes of materials, with minimal sample consumption, using routinely available devices. 1 Sentence summary Serial nano-beam electron diffraction (SerialNED) enables high-throughput, low dose protein crystallography from sub-micron crystals at conventional S/TEM microscopes.

We report on a method of fabricating lanthanum hexaboride (LaB6) cold field emission tips with sub-100-nm apices by using a combination of electrochemical etching and focused ion beam milling. The primary advantage of combining the two methods is rapid fabrication while maintaining reproducibility. The LaB6 tips have low work functions and high mechanical stabilities and are chemically inert to residual gases. Field emission characterization was performed on three tips, with apex sizes of 15, 85, and 80 nm yielding 10 nA cold field emission currents at 0.76, 3.9, and 3.6 kV extraction potentials, respectively. All three tips showed excellent emission current stability for periods exceeding 30 min in a 5 × 10−9 mbar vacuum.