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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

X-ray crystallographic studies on the hydrogen isotope effects of green fluorescent protein at sub-ångström resolutions.

Hydrogen atoms are critical to the nature and properties of proteins, and thus deuteration has the potential to influence protein function. In fact, it has been reported that some deuterated proteins show different physical and chemical properties to their protiated counterparts. Consequently, it is important to investigate protonation states around the active site when using deuterated proteins. Here, hydrogen isotope effects on the S65T/F99S/M153T/V163A variant of green fluorescent protein (GFP), in which the deprotonated B form is dominant at pH 8.5, were investigated. The pH/pD dependence of the absorption and fluorescence spectra indicates that the protonation state of the chromophore is the same in protiated GFP in H2O and protiated GFP in D2O at pH/pD 8.5, while the pKa of the chromophore became higher in D2O. Indeed, X-ray crystallographic analyses at sub-ångström resolution revealed no apparent changes in the protonation state of the chromophore between the two samples. However, detailed comparisons of the hydrogen OMIT maps revealed that the protonation state of His148 in the vicinity of the chromophore differed between the two samples. This indicates that protonation states around the active site should be carefully adjusted to be the same as those of the protiated protein when neutron crystallographic analyses of proteins are performed.

Subatomic resolution X-ray structures of green fluorescent protein.

Green fluorescent protein (GFP) is a light-emitting protein that does not require a prosthetic group for its fluorescent activity. As such, GFP has become indispensable as a molecular tool in molecular biology. Nonetheless, there has been no subatomic elucidation of the GFP structure owing to the structural polymorphism around the chromophore. Here, subatomic resolution X-ray structures of GFP without the structural polymorphism are reported. The positions of H atoms, hydrogen-bonding network patterns and accurate geometric parameters were determined for the two protonated forms. Compared with previously determined crystal structures and theoretically optimized structures, the anionic chromophores of the structures represent the authentic resonance state of GFP. In addition, charge-density analysis based on atoms-in-molecules theory and noncovalent interaction analysis highlight weak but substantial interactions between the chromophore and the protein environment. Considered with the derived chemical indicators, the lone pair-π interactions between the chromophore and Thr62 should play a sufficient role in maintaining the electronic state of the chromophore. These results not only reveal the fine structural features that are critical to understanding the properties of GFP, but also highlight the limitations of current quantum-chemical calculations.

Distribution of valence electrons of the flavin cofactor in NADH-cytochrome b5 reductase.

Flavin compounds such as flavin adenine dinucleotide (FAD), flavin mononucleotide and riboflavin make up the active centers in flavoproteins that facilitate various oxidoreductive processes. The fine structural features of the hydrogens and valence electrons of the flavin molecules in the protein environment are critical to the functions of the flavoproteins. However, information on these features cannot be obtained from conventional protein X-ray analyses at ordinary resolution. Here we report the charge density analysis of a flavoenzyme, NADH-cytochrome b5 reductase (b5R), at an ultra-high resolution of 0.78 Å. Valence electrons on the FAD cofactor as well as the peptide portion, which are clearly visualized even after the conventional refinement, are analyzed by the multipolar atomic model refinement. The topological analysis for the determined electron density reveals the valence electronic structure of the isoalloxazine ring of FAD and hydrogen-bonding interactions with the protein environment. The tetrahedral electronic distribution around the N5 atom of FAD in b5R is stabilized by hydrogen bonding with CαH of Tyr65 and amide-H of Thr66. The hydrogen bonding network leads to His49 composing the cytochrome b5-binding site via non-classical hydrogen bonds between N5 of FAD and CαH of Tyr65 and O of Tyr65 and CßH of His49.

Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer.

NADH-Cytochrome b5 reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5 (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD(+). Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.