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Crystallographic analysis relies on the scattering of quanta from arrays of atoms that populate a repeating lattice. While large crystals built of lattices that appear ideal are sought after by crystallographers, imperfections are the norm for molecular crystals. Additionally, advanced X-ray and electron diffraction techniques, used for crystallography, have opened the possibility of interrogating micro- and nanoscale crystals, with edges only millions or even thousands of molecules long. These crystals exist in a size regime that approximates the lower bounds for traditional models of crystal nonuniformity and imperfection. Accordingly, data generated by diffraction from both X-rays and electrons show increased complexity and are more challenging to conventionally model. New approaches in serial crystallography and spatially resolved electron diffraction mapping are changing this paradigm by better accounting for variability within and between crystals. The intersection of these methods presents an opportunity for a more comprehensive understanding of the structure and properties of nanocrystalline materials.

Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED.

Quantitative analysis of complex mixtures, including compounds having similar chemical properties, is demonstrated using an automatic and high throughput approach to microcrystal electron diffraction (MicroED). Compositional analysis of organic and inorganic compounds can be accurately executed without the need of diffraction standards. Additionally, with sufficient statistics, small amounts of compounds in mixtures can be reliably detected. These compounds can be distinguished by their crystal structure properties prior to structure solution. In addition, if the crystals are of good quality, the crystal structures can be generated on the fly, providing a complete analysis of the sample. MicroED is an effective method for analyzing the structural properties of sub-micron crystals, which are frequently found in small-molecule powders. By developing and using an automatic and high throughput approach to MicroED, and with the use of SerialEM for data collection, data from thousands of crystals allow sufficient statistics to detect even small amounts of compounds reliably.

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus (HCV) infection. Its structures have been elusive to the public until recently when one of the crystal forms is solved by microcrystal electron diffraction (MicroED). In this work, the MicroED structures of two distinct polymorphic crystal forms of paritaprevir are reported from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropyl sulfonamide and methyl pyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the HCV non-structural 3/4A (NS3/4A) serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acyl sulfonamide inhibitors to the HCV NS3/4A serine protease. In addition, this also demonstrates the opportunity to derive different polymorphs and distinct macrocycle conformations from the same experiments using MicroED.

Plants are able to produce various types of crystals through metabolic processes, serving functions ranging from herbivore deterrence to photosynthetic efficiency. However, the structural analysis of these crystals has remained challenging due to their small and often imperfect nature, which renders traditional X-ray diffraction techniques unsuitable. This study explores the use of Microcrystal Electron Diffraction (microED) as a novel method for the structural analysis of plant-derived microcrystals, focusing on Armeria maritima (Milld.), a halophytic plant known for its biomineralisation capabilities. In this study, A. maritima plants were cultivated under controlled laboratory conditions with exposure to cadmium and thallium to induce the formation of crystalline deposits on their leaf surfaces. These deposits were analysed using microED, revealing the presence of sodium chloride (halite), sodium sulphate (thénardite), and calcium sulphate dihydrate (gypsum). Our findings highlight the potential of microED as a versatile tool in plant science, capable of providing detailed structural insights into biomineralisation processes, even from minimal and imperfect crystalline samples. The application of microED in this context not only advances the present understanding of A. maritima's adaptation to saline environments but also opens new avenues for exploring the structural chemistry of biomineralisation in other plant species. Our study advocates for the broader adoption of microED in botanical research, especially when dealing with challenging crystallographic problems. [ABSTRACT FROM AUTHOR]

Eukaryotic TIR (Toll/interleukin‐1 receptor protein) domains signal via TIR–TIR interactions, either by self‐association or by interaction with other TIR domains. In mammals, TIR domains are found in Toll‐like receptors (TLRs) and cytoplasmic adaptor proteins involved in pro‐inflammatory signaling. Previous work revealed that the MAL TIR domain (MALTIR) nucleates the assembly of MyD88TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88TIR assembly has previously been solved, revealing a two‐stranded higher‐order assembly of TIR domains. In this work, it is demonstrated that the TIR domain of TLR2, which is reported to signal as a heterodimer with either TLR1 or TLR6, induces the formation of crystalline higher‐order assemblies of MyD88TIRin vitro, whereas TLR1TIR and TLR6TIR do not. Using an improved data‐collection protocol, the MicroED structure of TLR2TIR‐induced MyD88TIR microcrystals was determined at a higher resolution (2.85 Å) and with higher completeness (89%) compared with the previous structure of the MALTIR‐induced MyD88TIR assembly. Both assemblies exhibit conformational differences in several areas that are important for signaling (for example the BB loop and CD loop) compared with their monomeric structures. These data suggest that TLR2TIR and MALTIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88TIR assemblies unidirectionally. [ABSTRACT FROM AUTHOR]

Although COF-300 is often used as an example to study the synthesis and structure of (3D) covalent organic frameworks (COFs), knowledge of the underlying synthetic processes is still fragmented. Here, an optimized synthetic procedure based on a combination of linker protection and modulation was applied. Using this approach, the influence of time and temperature on the synthesis of COF-300 was studied. Synthesis times that were too short produced materials with limited crystallinity and porosity, lacking the typical pore flexibility associated with COF-300. On the other hand, synthesis times that were too long could be characterized by loss of crystallinity and pore order by degradation of the tetrakis(4-aminophenyl)methane (TAM) linker used. The presence of the degradation product was confirmed by visual inspection, Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). As TAM is by far the most popular linker for the synthesis of 3D COFs, this degradation process might be one of the reasons why the development of 3D COFs is still lagging compared with 2D COFs. However, COF crystals obtained via an optimized procedure could be structurally probed using 3D electron diffraction (3DED). The 3DED analysis resulted in a full structure determination of COF-300 at atomic resolution with satisfying data parameters. Comparison of our 3DED-derived structural model with previously reported single-crystal X-ray diffraction data for this material, as well as parameters derived from the Cambridge Structural Database, demonstrates the high accuracy of the 3DED method for structure determination. This validation might accelerate the exploitation of 3DED as a structure determination technique for COFs and other porous materials.

A series of events underscoring the significant advancements in micro-crystallization and in vivo crystallography were held during the 26th IUCr Congress in Melbourne, positioning microcrystallography as a pivotal field within structural biology. Through collaborative discussions and the sharing of innovative methodologies, these sessions outlined frontier approaches in macromolecular crystallography. This review provides an overview of this rapidly moving field in light of the rich dialogues and forward-thinking proposals explored during the congress workshop and microsymposium. These advances in microcrystallography shed light on the potential to reshape current research paradigms and enhance our comprehension of biological mechanisms at the molecular scale.

3D electron diffraction (3D ED), or microcrystal electron diffraction (MicroED), has become an alternative technique for determining the high‐resolution crystal structures of compounds from sub‐micron‐sized crystals. Here, we considered l‐alanine, α‐glycine and urea, which are known to form good‐quality crystals, and collected high‐resolution 3D ED data on our in‐house TEM instrument. In this study, we present a comparison of independent atom model (IAM) and transferable aspherical atom model (TAAM) kinematical refinement against experimental and simulated data. TAAM refinement on both experimental and simulated data clearly improves the model fitting statistics (R factors and residual electrostatic potential) compared to IAM refinement. This shows that TAAM better represents the experimental electrostatic potential of organic crystals than IAM. Furthermore, we compared the geometrical parameters and atomic displacement parameters (ADPs) resulting from the experimental refinements with the simulated refinements, with the periodic density functional theory (DFT) calculations and with published X‐ray and neutron crystal structures. The TAAM refinements on the 3D ED data did not improve the accuracy of the bond lengths between the non‐H atoms. The experimental 3D ED data provided more accurate H‐atom positions than the IAM refinements on the X‐ray diffraction data. The IAM refinements against 3D ED data had a tendency to lead to slightly longer X—H bond lengths than TAAM, but the difference was statistically insignificant. Atomic displacement parameters were too large by tens of percent for l‐alanine and α‐glycine. Most probably, other unmodelled effects were causing this behaviour, such as radiation damage or dynamical scattering. [ABSTRACT FROM AUTHOR]

New features in the dose estimation program RADDOSE‐3D are summarised. They include the facility to enter a diffraction intensity decay model which modifies the "Diffraction Weighted Dose" output from a "Fluence Weighted Dose" to a "Diffraction‐Decay Weighted Dose", a description of RADDOSE‐ED for use in electron diffraction experiments, where dose is historically quoted in electrons/Å2 rather than in gray (Gy), and finally the development of a RADDOSE‐3D GUI, enabling easy access to all the options available in the program. [ABSTRACT FROM AUTHOR]

We report on the latest advancements in Microcrystal Electron Diffraction (3D ED/MicroED), as discussed during a symposium at the National Center for CryoEM Access and Training housed at the New York Structural Biology Center. This snapshot describes cutting‐edge developments in various facets of the field and identifies potential avenues for continued progress. Key sections discuss instrumentation access, research applications for small molecules and biomacromolecules, data collection hardware and software, data reduction software, and finally reporting and validation. 3D ED/MicroED is still early in its wide adoption by the structural science community with ample opportunities for expansion, growth, and innovation. [ABSTRACT FROM AUTHOR]

Photochemical reactions are powerful tools for synthesizing organic molecules. The input of energy provided by light offers a means to produce strained and unique molecules that cannot be assembled using thermal protocols, allowing for the production of immense molecular complexity in a single chemical step. Furthermore, unlike thermal reactions, photochemical reactions do not require active reagents such as acids, bases, metals, or enzymes. Photochemical reactions play a central role in green chemistry. This article reports the isolation and structure determination of four new compounds (1–4) from the photoreaction products of the Polyozellus multiplex MeOH ext. The structures of the new compounds were elucidated using MS, IR, comprehensive NMR measurements and microED. The four compounds were formed by deacetylation of polyozellin, the main secondary metabolite of P. multiplex, and addition of singlet oxygen generated by sunlight. To develop drugs for treating Alzheimer's disease (AD) on the basis of the amyloid cascade hypothesis, the compounds (1–4) obtained by photoreaction were evaluated for BACE1 inhibitory activity. The hydrolysates (5 and 6) of polyozellin, the main secondary metabolites of P. multiplex, were also evaluated. The photoreaction products (3 and 4) and hydrolysates (5 and 6) of polyozellin showed BACE1 inhibitory activity (IC50: 2.2, 16.4, 23.3, and 5.3 μM, respectively). [ABSTRACT FROM AUTHOR]

The cryo-electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) was initially described in 2013 and has recently gained attention as an emerging technique for research in drug discovery. As compared to other methods in structural biology, MicroED provides many advantages deriving from the use of nanocrystalline material for the investigations. Here, we review the recent advancements in the field of MicroED and show important examples of small molecule, peptide and protein structures that has contributed to the current development of this method as an important tool for drug discovery.

Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.

X‐ray and electron diffraction methods independently identify the S‐enantiomer of Berkecoumarin [systematic name: (S)‐8‐hydroxy‐3‐(2‐hydroxypropyl)‐6‐methoxy‐2H‐chromen‐2‐one]. Isolated from Berkeley Pit Lake Penicillium sp., Berkecoumarin is a natural product with a light‐atom composition (C13H14O5) that challenges in‐house absolute structure determination by anomalous scattering. This study further demonstrates the utility of dynamical refinement of electron‐diffraction data for absolute structure determination. [ABSTRACT FROM AUTHOR]

Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED.

Plants are able to produce various types of crystals through metabolic processes, serving functions ranging from herbivore deterrence to photosynthetic efficiency. However, the structural analysis of these crystals has remained challenging due to their small and often imperfect nature, which renders traditional X-ray diffraction techniques unsuitable. This study explores the use of Microcrystal Electron Diffraction (microED) as a novel method for the structural analysis of plant-derived microcrystals, focusing on Armeria maritima (Milld.), a halophytic plant known for its biomineralisation capabilities. In this study, A. maritima plants were cultivated under controlled laboratory conditions with exposure to cadmium and thallium to induce the formation of crystalline deposits on their leaf surfaces. These deposits were analysed using microED, revealing the presence of sodium chloride (halite), sodium sulphate (thénardite), and calcium sulphate dihydrate (gypsum). Our findings highlight the potential of microED as a versatile tool in plant science, capable of providing detailed structural insights into biomineralisation processes, even from minimal and imperfect crystalline samples. The application of microED in this context not only advances the present understanding of A. maritima’s adaptation to saline environments but also opens new avenues for exploring the structural chemistry of biomineralisation in other plant species. Our study advocates for the broader adoption of microED in botanical research, especially when dealing with challenging crystallographic problems.

Electron crystallography has a storied history which rivals that of its more established X-ray-enabled counterpart. Recent advances in data collection and analysis have sparked a renaissance in the field, opening a new chapter for this venerable technique. Burgeoning interest in electron crystallography has spawned innovative methods described by various interchangeable labels (3D ED, MicroED, cRED, etc.). This Review covers concepts and findings relevant to the practicing crystallographer, with an emphasis on experiments aimed at using electron diffraction to elucidate the atomic structure of three-dimensional molecular crystals.

Cryoelectron microscopy (cryo-EM) methods have made meaningful contributions in a wide variety of scientific research fields. In structural biology, cryo-EM routinely elucidates molecular structure from isolated biological macromolecular complexes or in a cellular context by harnessing the high-resolution power of the electron in order to image samples in a frozen, hydrated environment. For structural chemistry, the cryo-EM method popularly known as microcrystal electron diffraction (MicroED) has facilitated atomic structure generation of peptides and small molecules from their three-dimensional crystal forms. As cryo-EM has grown from an emerging technology, it has undergone modernization to enable multimodal transmission electron microscopy (TEM) techniques becoming more routine, reproducible, and accessible to accelerate research across multiple disciplines. We review recent advances in modern cryo-EM and assess how they are contributing to the future of the field with an eye to the past. [Display omitted] In this review, de la Cruz and Eng discuss major developments in single-particle analysis, electron tomography, and microcrystal electron diffraction to show how cryo-EM has matured to become an ever-improving technology for visualizing molecular structure. [ABSTRACT FROM AUTHOR]

The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A2A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.

In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.

Three-dimensional electron diffraction (3D ED) is a measurement and analysis technique in transmission electron microscopy that is used for determining atomic structures from small crystals. Diverse targets such as proteins, polypeptides, and organic compounds, whose crystals exist in aqueous solutions and organic solvents, or as dried powders, can be studied with 3D ED. We have been involved in the development of this technique, which can now rapidly process a large number of data collected through AI control, enabling efficient structure determination. Here, we introduce this method and describe our recent results. These include the structures and pathogenic mechanisms of wild-type and mutant polypeptides associated with the debilitating disease amyotrophic lateral sclerosis (ALS), the double helical structure of nanographene promoting nanofiber formation, and the structural properties of an organic semiconductor containing disordered regions. We also discuss the limitations and prospects of 3D ED compared to microcrystallography with X-ray free electron lasers. [Display omitted] In this review, Yonekura et al. describe past and recent developments in the field of electron 3D crystallography and introduce their related work studying the pathogenic mechanisms of ALS and functional properties of organic materials. They also discuss the limitations and prospects of 3D ED compared to microcrystallography at XFELs. [ABSTRACT FROM AUTHOR]

In this study, we synthesized benzodioxol carboxamide derivatives and investigated their antidiabetic potential. The synthesized compounds (Ia-Ic and IIa-IId) underwent characterization via HRMS, 1H-, 13CAPT-NMR, and MicroED. Their efficacy against α-amylase was assessed in vitro, while MTS assays were employed to gauge cytotoxicity across cancer and normal cell lines. Additionally, the antidiabetic impact of compound IIc was evaluated in vivo using a streptozotocin-induced diabetic mice model. Notably, IIa and IIc displayed potent α-amylase inhibition (IC50 values of 0.85 and 0.68 µM, respectively) while exhibiting a negligible effect on the Hek293t normal cell line (IC50 > 150 µM), suggesting their safety. Compound IId demonstrated significant activity against four cancer cell lines (26–65 µM). In vivo experiments revealed that five doses of IIc substantially reduced mice blood glucose levels from 252.2 mg/dL to 173.8 mg/dL in contrast to the control group. The compelling in vitro anticancer efficacy of IIc and its safety for normal cells underscores the need for further in vivo assessment of this promising compound. This research highlights the potential of benzodioxol derivatives as candidates for the future development of synthetic antidiabetic drugs. [ABSTRACT FROM AUTHOR]

The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

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. [ABSTRACT FROM AUTHOR]

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

Establishing the absolute configuration of chiral active pharmaceutical ingredients (APIs) is of great importance. Single crystal X‐ray diffraction (scXRD) has traditionally been the method of choice for such analysis, but scXRD requires the growth of large crystals, which can be challenging. Here, we present a method for determining absolute configuration that does not rely on the growth of large crystals. By examining microcrystals formed with chiral probes (small chiral compounds such as amino acids), absolute configuration can be unambiguously determined by microcrystal electron diffraction (MicroED). Our streamlined method employs three steps: (1) virtual screening to identify promising chiral probes, (2) experimental cocrystal screening and (3) structure determination by MicroED and absolute configuration assignment. We successfully applied this method to analyze two chiral API molecules currently on the market for which scXRD was not used to determine absolute configuration. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.

Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.

Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.

The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.

Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.

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. [ABSTRACT FROM AUTHOR]

Aragon et al. [Acta Cryst. (2024), C80, 179–189], by reporting the discussion and the final conclusions of a round table held during a symposium at the National Center for CryoEM Access and Training, well describe all the advances that have been made for the application of 3D ED/MicroED to pharmaceutical and macromolecular nanocrystals and propose possible future scenarios. [ABSTRACT FROM AUTHOR]

This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.

In this study, we synthesized benzodioxol carboxamide derivatives and investigated their antidiabetic potential. The synthesized compounds (Ia-Ic and IIa-IId) underwent characterization via HRMS, 1H-, 13CAPT-NMR, and MicroED. Their efficacy against α-amylase was assessed in vitro, while MTS assays were employed to gauge cytotoxicity across cancer and normal cell lines. Additionally, the antidiabetic impact of compound IIc was evaluated in vivo using a streptozotocin-induced diabetic mice model. Notably, IIa and IIc displayed potent α-amylase inhibition (IC50 values of 0.85 and 0.68 µM, respectively) while exhibiting a negligible effect on the Hek293t normal cell line (IC50 > 150 µM), suggesting their safety. Compound IId demonstrated significant activity against four cancer cell lines (26–65 µM). In vivo experiments revealed that five doses of IIc substantially reduced mice blood glucose levels from 252.2 mg/dL to 173.8 mg/dL in contrast to the control group. The compelling in vitro anticancer efficacy of IIc and its safety for normal cells underscores the need for further in vivo assessment of this promising compound. This research highlights the potential of benzodioxol derivatives as candidates for the future development of synthetic antidiabetic drugs.

Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.

Sequence variation in the b2α2 loop, residues 165-175 of the mammalian prion protein (PrP), influences its structure. To better understand the consequences of sequence variation in this region of the protein, we biochemically and biophysically interrogate natural and artificial sequence variants of the b2α2 loop of mammalian PrP. Using microcrystal electron diffraction (MicroED), we determine atomic resolution structures of segments encompassing residues 168-176 from the b2α2 loop of PrP with sequences corresponding to human, mouse/cow, bank vole/hamster, rabbit/pig/guinea pig, and naked mole rat (elk-T174S) b2α2 loops, as well as synthetic b2α2 loop sequences. This collection of structures presents two dominant amyloid packing polymorphisms. In the first polymorph, denoted “clasped”, side chains within a sheet form polar clasps by facing each other on the same strand, exemplified by the mouse/cow, human, and bank vole/hamster sequences. Because its stability is derived from within a strand and through polar ladders within a sheet, the sequence requirements for the mating strand are less restrictive. A second polymorph, denoted “interdigitated,” has sidechains interdigitate across mating sheets, exemplified by the elk, naked mole rat (elk T174S), and rabbit sequences. The two types of packing present distinct networks of stabilizing hydrogen bonds. The identity of residue 174 appears to strongly influence the packing adopted in these peptides, but consideration of the overall sequence of a given segment is needed to understand the stability of its assemblies. Incorporation of these b2α2 loop sequences into an 85 residue recombinant segment encoding wild-type bank vole PrP94-178 demonstrates that even single residue substitutions could impact fibril morphology as evaluated by negative stain electron microscopy. This is in line with recent findings supporting the accessibility of different structural geometries by varied mammalian prion sequences, and indicates that sequence-specific polymorphisms may be influenced by residues in the b2α2 loop. [ABSTRACT FROM AUTHOR]

Sequence variation in the β2α2 loop, residues 165-175 of the mammalian prion protein (PrP), influences its structure. To better understand the consequences of sequence variation in this region of the protein, we biochemically and biophysically interrogate natural and artificial sequence variants of the β2α2 loop of mammalian PrP. Using microcrystal electron diffraction (MicroED), we determine atomic resolution structures of segments encompassing residues 168-176 from the β2α2 loop of PrP with sequences corresponding to human, mouse/cow, bank vole/hamster, rabbit/pig/guinea pig, and naked mole rat (elk-T174S) β2α2 loops, as well as synthetic β2α2 loop sequences. This collection of structures presents two dominant amyloid packing polymorphisms. In the first polymorph, denoted “clasped”, side chains within a sheet form polar clasps by facing each other on the same strand, exemplified by the mouse/cow, human, and bank vole/hamster sequences. Because its stability is derived from within a strand and through polar ladders within a sheet, the sequence requirements for the mating strand are less restrictive. A second polymorph, denoted “interdigitated,” has sidechains interdigitate across mating sheets, exemplified by the elk, naked mole rat (elk T174S), and rabbit sequences. The two types of packing present distinct networks of stabilizing hydrogen bonds. The identity of residue 174 appears to strongly influence the packing adopted in these peptides, but consideration of the overall sequence of a given segment is needed to understand the stability of its assemblies. Incorporation of these β2α2 loop sequences into an 85 residue recombinant segment encoding wild-type bank vole PrP94–178 demonstrates that even single residue substitutions could impact fibril morphology as evaluated by negative stain electron microscopy. This is in line with recent findings supporting the accessibility of different structural geometries by varied mammalian prion sequences, and indicates that sequence-specific polymorphisms may be influenced by residues in the β2α2 loop.

Electron radiation damage to macromolecules is an inevitable resolution limit factor in all major structural determination applications using cryo-electron microscopy (cryo-EM). Single particle analysis (SPA) and micro-crystal electron diffraction (MicroED) have been employed to assess radiation damage with a variety of protein complexes. Although radiation induced sidechain density loss and resolution decay were observed by both methods, the minimum dose of electron irradiation reducing high-resolution limit reported by SPA is more than ten folds higher than measured by MicroED using the conventional dose concept, and there is a gap between the attained resolutions assessed by these two methods. We compared and analyzed these two approaches side-by-side in detail from several aspects to identify some crucial determinants and to explain this discrepancy. Probability of a high energy electron being inelastically scattered by a macromolecule is proportional to number of layers of the molecules in its transmission path. As a result, the same electron dose could induce much more site-specific damage to macromolecules in 3D protein crystal than single particle samples. Major differences in data collection and processing scheme are the key factors to different levels of sensitivity to radiation damage at high resolution between the two methods. High resolution electron diffraction in MicroED dataset is very sensitive to global damage to 3D protein crystals with low dose accumulation, and its intensity attenuation rates at atomic resolution shell could be applied for estimating ratio of damaged and total selected single particles for SPA. More in-depth systematically radiation damage assessments using SPA and MicroED will benefit all applications of cryo-EM, especially cellular structure analysis by tomography.

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.

Once the optical, electronic, or photocatalytic properties of a semiconductor are set by adjusting composition, crystal phase, and morphology, one cannot change them anymore, respectively, on demand. Materials enabling postsynthetic and reversible switching of features such as absorption coefficient, bandgap, or charge carrier dynamics are highly desired. Hybrid perovskites facilitate exceptional possibilities for progress in the field of smart semiconductors because active organic molecules become an integral constituent of the crystalline structure. This paper reports the integration of ferrocene ligands into semiconducting 2D phases based on lead bromide. The complex crystal structures of the resulting, novel ferrovskite (≃ ferrocene perovskite) phases are determined by 3D electron diffraction. The ferrocene ligands exhibit strong structure‐directing effects on the 2D hybrid phases, which is why the formation of exotic types of face‐ and edge‐sharing lead bromide octahedra is observed. The bandgap of the materials ranges from 3.06 up to 3.51 eV, depending on the connectivity of the octahedra. By deploying the redox features of ferrocene, one can create defect states or even a defect band leading to control over the direction of exciton migration and energy transport in the semiconductor, enabling fluorescence via indirect to direct gap transition. [ABSTRACT FROM AUTHOR]

Three‐dimensional electron diffraction (3D ED)/microcrystal electron diffraction (MicroED) techniques are gaining in popularity. However, the data processing often does not fit existing graphical user interface software, instead requiring the use of the terminal or scripting. Scipion‐ED, described in this article, provides a graphical user interface and extendable framework for processing of 3D ED/MicroED data. An illustrative project is described, in which multiple 3D ED/MicroED data sets collected on tetragonal lysozyme were processed with DIALS through the Scipion‐ED interface. The ability to resolve unmodelled features in the electrostatic potential map was compared between three strategies for merging data sets. [ABSTRACT FROM AUTHOR]

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.