ALS mutations in the TIA-1 prion-like domain trigger highly condensed pathogenic structures.

T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing ß-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity.

Comprehensive Application of XFEL Microcrystallography for Challenging Targets in Various Organic Compounds.

There is a growing demand for structure determination from small crystals, and the three-dimensional electron diffraction (3D ED) technique can be employed for this purpose. However, 3D ED has certain limitations related to the crystal thickness and data quality. We here present the application of serial X-ray crystallography (SX) with X-ray free electron lasers (XFELs) to small (a few µm or less) and thin (a few hundred nm or less) crystals of novel compounds dispersed on a substrate. For XFEL exposures, two-dimensional (2D) scanning of the substrate coupled with rotation enables highly efficient data collection. The recorded patterns can be successfully indexed using lattice parameters obtained through 3D ED. This approach is especially effective for challenging targets, including pharmaceuticals and organic materials that form preferentially oriented flat crystals in low-symmetry space groups. Some of these crystals have been difficult to solve or have yielded incomplete solutions using 3D ED. Our extensive analyses confirmed the superior quality of the SX data regardless of crystal orientations. Additionally, 2D scanning with XFEL pulses gives an overall distribution of the samples on the substrate, which can be useful for evaluating the properties of crystal grains and the quality of layered crystals. Therefore, this study demonstrates that XFEL crystallography has become a powerful tool for conducting structure studies of small crystals of organic compounds.

Double-Helix Supramolecular Nanofibers Assembled from Negatively Curved Nanographenes.

The layered structures of graphite and related nanographene molecules play key roles in their physical and electronic functions. However, the stacking modes of negatively curved nanographenes remain unclear, owing to the lack of suitable nanographene molecules. Herein, we report the synthesis and one-dimensional supramolecular self-assembly of negatively curved nanographenes without any assembly-assisting substituents. This curved nanographene self-assembles in various organic solvents and acts as an efficient gelator. The formation of nanofibers was confirmed by microscopic measurements, and an unprecedented double-helix assembly by continuous π-π stacking was uncovered by three-dimensional electron crystallography. This work not only reports the discovery of an all-sp2-carbon supramolecular π-organogelator with negative curvature but also demonstrates the power of three-dimensional electron crystallography for the structural determination of submicrometer-sized molecular alignment.

Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus.

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

Collecting large datasets of rotational electron diffraction with ParallEM and SerialEM.

A semi-automated protocol has been developed for rotational data collection of electron diffraction patterns by combined use of SerialEM and ParallEM, where SerialEM is used for positioning of sample crystals and ParallEM for rotational data collection. ParallEM calls standard camera control software through an AutoIt script, which adapts to software operational changes and to new GUI programs guiding other cameras. Development included periodic flashing and pausing of data collection during overnight or day-long recording with a cold field-emission beam. The protocol proved to be efficient and accurate in data collection of large-scale rotational series from two JEOL electron microscopes, a general-purpose JEM-2100 and a high-end CRYO ARM 300. Efficiency resulted from simpler steps and task specialization. It is possible to collect 12-20 rotational series from ~-68° to ~68° at a rotation speed of 1°/s in one hour without human supervision.

A new cryo-EM system for electron 3D crystallography by eEFD.

A new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, filters out energy-loss electrons through the sample with an in-column energy filter, and allows rotational data collection on a fast camera. It also possesses motorized cryo-sample loading and automated liquid-nitrogen filling for cooling of multiple samples. To facilitate its use, we developed GUI programs for efficient operation and accurate structure analysis. Here we report on the performance of the system and first results for thin 3D crystals of the protein complexes, catalase and membrane protein complex ExbBD. Data quality is remarkably improved with this approach, which we name eEFD (electron energy-filtered diffraction of 3D crystals), compared with those collected at 200 kV without energy filtration. Key advances include precise control of the microscope and recordings of lens fluctuations, which the programs process and respond to. We also discuss the merits of higher-energy electrons and filtration of energy-loss electrons in electron 3D crystallography.

A new cryo-EM system for single particle analysis.

A new cryo-EM system has been investigated for single particle analysis of protein structures. The system provides parallel illumination of a highly-coherent 300 kV electron beam from a cold-field emission gun, and boosts image contrast with an in-column energy filter and a hole-free phase plate. It includes motorized cryo-sample loading and automated liquid-nitrogen filling for cooling multiple samples. In this study, we describe gun and electron beam characteristics, and demonstrate the suitability of this system for single particle reconstructions. The performance of the system is tested on two examples, a spherical virus and apoferritin. GUI programs have also been developed to control and monitor the system for correct illumination, imaging with less ellipticity and steady magnification, and timing of flashing and liquid-nitrogen filling. These programs are especially useful for efficient application of the system to single particle cryo-EM.

Applications and limitations of electron 3D crystallography.

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.

Measurement of charges and chemical bonding in a cryo-EM structure.

Hydrogen bonding, bond polarity, and charges in protein molecules play critical roles in the stabilization of protein structures, as well as affecting their functions such as enzymatic catalysis, electron transfer, and ligand binding. These effects can potentially be measured in Coulomb potentials using cryogenic electron microscopy (cryo-EM). We here present charges and bond properties of hydrogen in a sub-1.2 Å resolution structure of a protein complex, apoferritin, by single-particle cryo-EM. A weighted difference map reveals positive densities for most hydrogen atoms in the core region of the complex, while negative densities around acidic amino-acid side chains are likely related to negative charges. The former positive densities identify the amino- and oxo-termini of asparagine and glutamine side chains. The latter observations were verified by spatial-resolution selection and a dose-dependent frame series. The average position of the hydrogen densities depends on the parent bonded-atom type, and this is validated by the estimated level of the standard uncertainties in the bond lengths.

Structural resolution of a small organic molecule by serial X-ray free-electron laser and electron crystallography.

Structure analysis of small crystals is important in areas ranging from synthetic organic chemistry to pharmaceutical and material sciences, as many compounds do not yield large crystals. Here we present the detailed characterization of the structure of an organic molecule, rhodamine-6G, determined at a resolution of 0.82 Å by an X-ray free-electron laser (XFEL). Direct comparison of this structure with that obtained by electron crystallography from the same sample batch of microcrystals shows that both methods can accurately distinguish the position of some of the hydrogen atoms, depending on the type of chemical bond in which they are involved. Variations in the distances measured by XFEL and electron diffraction reflect the expected differences in X-ray and electron scatterings. The reliability for atomic coordinates was found to be better with XFEL, but the electron beam showed a higher sensitivity to charges.

Enantioselectivity of discretized helical supramolecule consisting of achiral cobalt phthalocyanines via chiral-induced spin selectivity effect.

Enantioselectivity of helical aggregation is conventionally directed either by its homochiral ingredients or by introduction of chiral catalysis. The fundamental question, then, is whether helical aggregation that consists only of achiral components can obtain enantioselectivity in the absence of chiral catalysis. Here, by exploiting enantiospecific interaction due to chiral-induced spin selectivity (CISS) that has been known to work to enantio-separate a racemic mixture of chiral molecules, we demonstrate the enantioselectivity in the assembly of mesoscale helical supramolecules consisting of achiral cobalt phthalocyanines. The helical nature in our supramolecules is revealed to be mesoscopically incorporated by dislocation-induced discretized twists, unlike the case of chiral molecules whose chirality are determined microscopically by chemical bond. The relevance of CISS effect in the discretized helical supramolecules is further confirmed by the appearance of spin-polarized current through the system. These observations mean that the application of CISS-based enantioselectivity is no longer limited to systems with microscopic chirality but is expanded to the one with mesoscopic chirality.

Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation.

The bacterial flagellar motor is driven by an ion flux through a channel called MotAB in Escherichia coli or Salmonella and PomAB in Vibrio alginolyticus. PomAB is composed of two transmembrane (TM) components, PomA and PomB, and converts a sodium ion flux to rotation of the flagellum. Its homolog, MotAB, utilizes protons instead of sodium ions. PomB/MotB has a peptidoglycan (PG)-binding motif in the periplasmic domain, allowing it to function as the stator by being anchored to the PG layer. To generate torque, PomAB/MotAB is thought to undergo a conformational change triggered by the ion flux and to interact directly with FliG, a component of the rotor. Here, we present the first three-dimensional structure of this torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the TM and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a "plugged" conformation, which would prevent ions leaking through the channel. We propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators.

Characterization of the yeast telomere nucleoprotein core: Rap1 binds independently to each recognition site.

At the core of Saccharomyces cerevisiae telomeres is an array of tandem telomeric DNA repeats bound site-specifically by multiple Rap1 molecules. There, Rap1 orchestrates the binding of additional telomere-associated proteins and negatively regulates both telomere fusion and length homeostasis. Using electron microscopy, viscosity, and light scattering measurements, we show that purified Rap1 is a monomer in solution that adopts a ringlike or C shape with a central cavity. Rap1 could orchestrate telomere function by binding multiple telomere array sites through either cooperative or independent mechanisms. To determine the mechanism, we analyze the distribution of Rap1 monomers on defined telomeric DNA arrays. This analysis clearly indicates that Rap1 binds independently to each nonoverlapping site in an array, regardless of the spacing between sites, the total number of sites, the affinity of the sites for Rap1, and over a large concentration range. Previous experiments have not clearly separated the effects of affinity from repeat spacing on telomere function. We clarify these results by testing in vivo the function of defined telomere arrays containing the same Rap1 binding site separated by spacings that were previously defined as low or high activity. We find that Rap1 binding affinity in vitro correlates with the ability of telomeric repeat arrays to regulate telomere length in vivo. We suggest that Rap1 binding to multiple sites in a telomere array does not, by itself, promote formation of a more energetically stabile complex.

Corrigendum: Protein and Organic-Molecular Crystallography with 300kV Electrons on a Direct Electron Detector.

[This corrects the article DOI: 10.3389/fmolb.2020.612226.].

Machine learning-based real-time object locator/evaluator for cryo-EM data collection.

In cryo-electron microscopy (cryo-EM) data collection, locating a target object is error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation shows its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and in locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection. The proposed approach will advance high-throughput and accurate data collection of images and diffraction patterns with minimal human operation.

Advances in cryo-EM and ED with a cold-field emission beam and energy filtration -Refinements of the CRYO ARM 300 system in RIKEN SPring-8 center.

We have designed and evaluated a cryo-electron microscopy (cryo-EM) system for higher-resolution single particle analysis and high-precision electron 3D crystallography. The system comprises a JEOL CRYO ARM 300 electron microscope-the first machine of this model-and a direct detection device camera, a scintillator-coupled camera, GPU clusters connected with a camera control computer and software for automated-data collection and efficient and accurate operation. The microscope provides parallel illumination of a highly coherent 300-kV electron beam to a sample from a cold-field emission gun and filters out energy-loss electrons through the sample with an in-column energy filter. The gun and filter are highly effective in improving imaging and diffraction, respectively, and have provided high quality data since July 2018. We here report on the characteristics of the cryo-EM system, updates, our progress and future plan for running such cryo-EM machines in RIKEN SPring-8 Center.

Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy.

The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments in two distinct conformations, L- and R-type, for supercoiling. The X-ray crystal structure of a flagellin fragment lacking about 100 terminal residues revealed the protofilament structure, but the full filament structure is still essential for understanding the mechanism of supercoiling and polymerization. Here we report a complete atomic model of the R-type filament by electron cryomicroscopy. A density map obtained from image data up to 4 A resolution shows the feature of alpha-helical backbone and some large side chains. The atomic model built on the map reveals intricate molecular packing and an alpha-helical coiled coil formed by the terminal chains in the inner core of the filament, with its intersubunit hydrophobic interactions having an important role in stabilizing the filament.

Protein and Organic-Molecular Crystallography With 300kV Electrons on a Direct Electron Detector.

Electron 3D crystallography can reveal the atomic structure from undersized crystals of various samples owing to the strong scattering power of electrons. Here, a direct electron detector DE64 was tested for small and thin crystals of protein and an organic molecule using a JEOL CRYO ARM 300 electron microscope. The microscope is equipped with a cold-field emission gun operated at an accelerating voltage of 300 kV, quad condenser lenses for parallel illumination, an in-column energy filter, and a stable rotational goniometer stage. Rotational diffraction data were collected in an unsupervised manner from crystals of a heme-binding enzyme catalase and a representative organic semiconductor material Ph-BTBT-C10. The structures were determined by molecular replacement for catalase and by the direct method for Ph-BTBT-C10. The analyses demonstrate that the system works well for electron 3D crystallography of these molecules with less damaging, a smaller point spread, and less noise than using the conventional scintillator-coupled camera.

Apple latent spherical virus structure with stable capsid frame supports quasi-stable protrusions expediting genome release.

Picorna-like plant viruses are non-enveloped RNA spherical viruses of ~30 nm. Part of the survival of these viruses depends on their capsid being stable enough to harbour the viral genome and yet malleable enough to allow its release. However, molecular mechanisms remain obscure. Here, we report a structure of a picorna-like plant virus, apple latent spherical virus, at 2.87 Å resolution by single-particle cryo-electron microscopy (cryo-EM) with a cold-field emission beam. The cryo-EM map reveals a unique structure composed of three capsid proteins Vp25, Vp20, and Vp24. Strikingly Vp25 has a long N-terminal extension, which substantially stabilises the capsid frame of Vp25 and Vp20 subunits. Cryo-EM images also resolve RNA genome leaking from a pentameric protrusion of Vp24 subunits. The structures and observations suggest that genome release occurs through occasional opening of the Vp24 subunits, possibly suppressed to a low frequency by the rigid frame of the other subunits.