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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.

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 (MAL TIR ) nucleates the assembly of MyD88 TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88 TIR 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 MyD88 TIR in vitro, whereas TLR1 TIR and TLR6 TIR do not. Using an improved data-collection protocol, the MicroED structure of TLR2 TIR -induced MyD88 TIR microcrystals was determined at a higher resolution (2.85 Å) and with higher completeness (89%) compared with the previous structure of the MAL TIR -induced MyD88 TIR 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 TLR2 TIR and MAL TIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88 TIR assemblies unidirectionally., (open access.)

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., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tamir Gonen reports financial support was provided by National Institute of Health. Tamir Gonen reports financial support was provided by Howard Hughes Medical Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

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., (open access.)

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., (open access.)

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/Å 2 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., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

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., (open access.)

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.

Parkinson's disease (PD) is a significant health issue because it gradually damages the nervous system. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors play a significant role in the development of PD. The current investigation employed hybrid benzodioxole-propanamide (BDZ-P) compounds to get information on AMPA receptors, analyze their biochemical and biophysical properties, and assess their neuroprotective effects. Examining the biophysical characteristics of all the subunits of the AMPA receptor offers insights into the impact of BDZ-P on the desensitization and deactivation rate. It demonstrates a partial improvement in the locomotor capacities in a mouse model of Parkinson's disease. In addition, the in vivo experiment assessed the locomotor activity by utilizing the open-field test. Our findings demonstrated that BDZ-P7 stands out with its remarkable potency, inhibiting the GluA2 subunit nearly 8-fold with an IC 50 of 3.03 μM, GluA1/2 by 7.5-fold with an IC 50 of 3.14 μM, GluA2/3 by nearly 7-fold with an IC 50 of 3.19 μM, and GluA1 by 6.5-fold with an IC 50 of 3.2 μM, significantly impacting the desensitization and deactivation rate of the AMPA receptor. BDZ-P7 showed an in vivo impact of partially reinstating locomotor abilities in a mouse model of PD. The results above suggest that the BDZ-P7 compounds show great promise as top contenders for the development of novel neuroprotective therapies.

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.

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 (IC 50 : 2.2, 16.4, 23.3, and 5.3 μM, respectively)., (© 2024. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

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., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

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., (open access.)

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., (open access.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C- termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.

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., (© 2024 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)

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 (C 13 H 14 O 5 ) 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., (open access.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Electron diffraction from three dimensional crystals, as a technique for solving molecular structures, is rapidly increasing in popularity. The development of methodology and software has borrowed, to great effect, from macromolecular X-ray crystallography. However, standardization lags behind the development of the technique, and practitioners are forced to work with inadequate data formats that are unable to capture a full description of their experiments. This creates obstacles that are increasingly difficult to overcome as experiments become ever faster and the need for data autoprocessing becomes more pressing. We present a data format standard based on best practice from macromolecular crystallography and demonstrate how the adoption of this standard enabled autoprocessing of datasets collected with a high-throughput detector system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The combination of high sensitivity and rapid readout makes it possible for electron-counting detectors to record cryogenic electron microscopy data faster and more accurately without increasing the number of electrons used for data collection. This is especially useful for MicroED of macromolecular crystals where the strength of the diffracted signal at high resolution is comparable to the surrounding background. The ability to decrease fluence also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. The major concern with electron-counting direct detectors lies at the low end of the resolution spectrum: their limited linear range makes strong low-resolution reflections susceptible to coincidence loss and careful data collection is required to avoid compromising data quality. Nevertheless, these cameras are increasingly deployed in cryo-EM facilities, and several have been successfully used for MicroED. Provided coincidence loss can be minimized, electron-counting detectors bring high potential rewards., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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, 1 H-, 13 CAPT-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 (IC 50 values of 0.85 and 0.68 µM, respectively) while exhibiting a negligible effect on the Hek293t normal cell line (IC 50 > 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.

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., (open access.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

As structural determination of protein complexes approaches atomic resolution, there is an increasing focus on conformational dynamics. Here we conceptualize the combination of two techniques which have become established in recent years: microcrystal electron diffraction and ultrafast electron microscopy. We show that the extremely low dose of pulsed photoemission still enables microED due to the strength of the electron bunching from diffraction of the protein crystals. Indeed, ultrafast electron diffraction experiments on protein crystals have already been demonstrated to be effective in measuring intermolecular forces in protein microcrystals. We discuss difficulties that may arise in the acquisition and processing of data and the overall feasibility of the experiment, paying specific attention to dose and signal-to-noise ratio. In doing so, we outline a detailed workflow that may be effective in minimizing the dose on the specimen. A series of model systems that would be good candidates for initial experiments is provided., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that the scattered electrons carry information about the charged state of atoms and provide relatively stronger contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for understanding protein structure and function, in particular for drug discovery. However, identification of individual hydrogen atom positions typically requires atomic resolution data, and has thus far remained elusive for macromolecular MicroED. Recently, we presented the ab initio structure of triclinic hen egg-white lysozyme at 0.87 Å resolution. The corresponding data were recorded under low exposure conditions using an electron-counting detector from thin crystalline lamellae. Here, using these subatomic resolution MicroED data, we identified over a third of all hydrogen atom positions based on strong difference peaks, and directly visualize hydrogen bonding interactions and the charged states of residues. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering the hydrogen atoms and hydrogen bonding interactions underlying protein structure and function., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Electron diffraction patterns unavoidably contain in-plane distortions introduced by electromagnetic lens systems. Geometric correction of different distortion types allows the accuracy of lattice parameter determination to be improved in 3D electron diffraction data., (© Tatiana Gorelik 2022.)

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 PrP 94-178 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., Competing Interests: JR is an equity stakeholder in Medstruc Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Glynn, Hernandez, Gallagher-Jones, Miao, Sigurdson and Rodriguez.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shi and Huang.)

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., (© Viktor E. G. Bengtsson et al. 2022.)

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., (© 2022 The Author(s).)

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal-bacterial symbiotic interactions., (© 2021 Wiley-VCH GmbH.)

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., (© Max T.B. Clabbers et al. 2022.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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 A 2A 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 A 2A 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., Competing Interests: The authors declare no competing interest.

Metabolomics has emerged as a powerful discipline to study complex biological systems from a small molecule perspective. The success of metabolomics hinges upon reliable annotations of spectral features obtained from MS and/or NMR. In spite of tremendous progress with regards to analytical instrumentation and computational tools, < 20% of spectral features are confidently identified in most untargeted metabolomics experiments. This article explores the integration of multiple analytical instruments such as UHPLC-MS/MS-SPE-NMR and the cryo-EM method MicroED to achieve large-scale and confident metabolite identifications in a higher-throughput manner. UHPLC-MS/MS-SPE allows for the simultaneous automated purification of metabolites followed by offline structure elucidation and structure validation by NMR and MicroED. Large-scale study of complex metabolomes such as that of the model plant legume Medicago truncatula can be achieved using an integrated UHPLC-MS/MS-SPE-NMR metabolomics platform. Additionally, recent developments in MicroED to study structures of small organic molecules have enabled faster, easier and precise structure determinations of metabolites. A MicroED small molecule structure elucidation workflow (e.g., crystal screening, sample preparation, data collection and data processing/structure determination) has been described. Ongoing MicroED methods development and its future scope related to structure elucidation of specialized metabolites and metabolomics are highlighted. The incorporation of MicroED with a UHPLC-MS/MS-SPE-NMR instrumental ensemble offers the potential to accelerate and achieve higher rates of metabolite identification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ghosh, Bu, Nannenga and Sumner.)

Three-dimensional electron diffraction crystallography (microED) can solve structures of sub-micrometer crystals, which are too small for single crystal X-ray crystallography. However, R factors for the microED-based structures are generally high because of dynamic scattering. That means R factor may not be reliable provided that kinetic analysis is used. Consequently, there remains ambiguity to locate hydrogens and to assign nuclei with close atomic numbers, like carbon, nitrogen, and oxygen. Herein, we employed microED and ssNMR dipolar-based experiments together with spin dynamics numerical simulations. The NMR dipolar-based experiments were 1 H- 14 N phase-modulated rotational-echo saturation-pulse double-resonance (PM-S-RESPDOR) and 1 H- 1 H selective recoupling of proton (SERP) experiments. The former examined the dephasing effect of a specific 1 H resonance under multiple 1 H- 14 N dipolar couplings. The latter examined the selective polarization transfer between a 1 H- 1 H pair. The structure was solved by microED and then validated by evaluating the agreement between experimental and calculated dipolar-based NMR results. As the measurements were performed on 1 H and 14 N, the method can be employed for natural abundance samples. Furthermore, the whole validation procedure was conducted at 293 K unlike widely used chemical shift calculation at 0 K using the GIPAW method. This combined method was demonstrated on monoclinic l-histidine.

The emerging field of microcrystal electron diffraction (MicroED) is of great interest to industrial researchers working in the drug discovery and drug development space. The promise of being able to routinely solve high-resolution crystal structures without the need to grow large crystals is very appealing. Despite MicroED's exciting potential, adoption across the pharmaceutical industry has been slow, primarily owing to a lack of access to specialized equipment and expertise. Here we present our experience building a small molecule MicroED service pipeline for members of the pharmaceutical industry. In the past year, we have examined more than fifty small molecule samples submitted by our clients, the majority of which have yielded data suitable for structure solution. We also detail our experience determining small molecule MicroED structures of pharmaceutical interest and offer some insights into the typical experimental outcomes. This experience has led us to conclude that small molecule MicroED adoption will continue to grow within the pharmaceutical industry where it is able to rapidly provide structures inaccessible by other methods., Competing Interests: JB, GS, AC, TG, SD, BR, TN, KL, MMy, NC, NP, PM, TW, MMs, CH, ST, CP, and BC are or have been employed by NanoImaging Services, a commercial supplier of electron microscopy services to the biopharmaceutical and biotechnology industries. BW is employed by the company Biogen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bruhn, Scapin, Cheng, Mercado, Waterman, Ganesh, Dallakyan, Read, Nieusma, Lucier, Mayer, Chiang, Poweleit, McGilvray, Wilson, Mashore, Hennessy, Thomson, Wang, Potter and Carragher.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bücker, Hogan-Lamarre and Miller.)

Aggregation-induced emission (AIE) phenomena have gained intense interest over the last decades because of its importance in solid-state emission. However, the elucidation of a working mechanism is difficult owing to the limited characterization methods on solid-state molecules, further complicated if dynamic structural changes occur. Here, a series of bis-arylacylhydrazones ( BAHs ) were synthesized, for which their AIE properties are only turned on by the reversible adsorption of water molecules. We used microcrystal electron diffraction (MicroED) to determine the molecular structures of two BAHs directly from bulk powders (without attempting to grow crystals) prepared in the absence or presence of water adsorption. This study reveals the unambiguous characterization of the dependence of crystal packing on the specific cocrystallization with hydrates. The structural analysis demonstrates that water molecules form strong hydrogen bonds with three neighboring BAH-1 , resulting in the almost complete planarization and restriction of the intramolecular rotation of the molecule. MicroED plays an important role in providing a decisive clue for the reversible polymorphism changes induced by the adsorption of water molecules, regulating emissive properties.

Mankind has long utilized Cannabis for diverse purposes. However, it has only been since the late 19th century that its individual cannabinoids began to be isolated, analyzed, and synthesized. By the mid-20th century it was discovered that many cannabinoids were asymmetric, with chirality often controlling their pharmacology. Increasingly accurate measurement and understanding of cannabinoid chirality will facilitate their synthesis and accelerate their medicinal applications., Competing Interests: No competing financial interests exist., (Copyright 2021, Mary Ann Liebert, Inc., publishers.)