X-Ray Free-Electron Lasers for the Structure and Dynamics of Macromolecules.

X-ray free-electron lasers provide femtosecond-duration pulses of hard X-rays with a peak brightness approximately one billion times greater than is available at synchrotron radiation facilities. One motivation for the development of such X-ray sources was the proposal to obtain structures of macromolecules, macromolecular complexes, and virus particles, without the need for crystallization, through diffraction measurements of single noncrystalline objects. Initial explorations of this idea and of outrunning radiation damage with femtosecond pulses led to the development of serial crystallography and the ability to obtain high-resolution structures of small crystals without the need for cryogenic cooling. This technique allows the understanding of conformational dynamics and enzymatics and the resolution of intermediate states in reactions over timescales of 100 fs to minutes. The promise of more photons per atom recorded in a diffraction pattern than electrons per atom contributing to an electron micrograph may enable diffraction measurements of single molecules, although challenges remain.

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Structural parasitology of the malaria parasite Plasmodium falciparum.

The difficulty of faithfully recapitulating malarial protein complexes in heterologous expression systems has long impeded structural study for much of the Plasmodium falciparum proteome. However, recent advances in single-particle cryo electron microscopy (cryoEM) now enable structure determination at atomic resolution with significantly reduced requirements for both sample quantity and purity. Combined with recent developments in gene editing, these advances open the door to structure determination and structural proteomics of macromolecular complexes enriched directly from P. falciparum parasites. Furthermore, the combination of cryoEM with the rapidly emerging use of in situ cryo electron tomography (cryoET) to directly visualize ultrastructures and protein complexes in the native cellular context will yield exciting new insights into the molecular machinery underpinning malaria parasite biology and pathogenesis.

Structure determination by cryoEM at 100 keV.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

Simcryocluster: a semantic similarity clustering method of cryo-EM images by adopting contrastive learning.

BACKGROUND: Cryo-electron microscopy (Cryo-EM) plays an increasingly important role in the determination of the three-dimensional (3D) structure of macromolecules. In order to achieve 3D reconstruction results close to atomic resolution, 2D single-particle image classification is not only conducive to single-particle selection, but also a key step that affects 3D reconstruction. The main task is to cluster and align 2D single-grain images into non-heterogeneous groups to obtain sharper single-grain images by averaging calculations. The main difficulties are that the cryo-EM single-particle image has a low signal-to-noise ratio (SNR), cannot manually label the data, and the projection direction is random and the distribution is unknown. Therefore, in the low SNR scenario, how to obtain the characteristic information of the effective particles, improve the clustering accuracy, and thus improve the reconstruction accuracy, is a key problem in the 2D image analysis of single particles of cryo-EM. RESULTS: Aiming at the above problems, we propose a learnable deep clustering method and a fast alignment weighted averaging method based on frequency domain space to effectively improve the class averaging results and improve the reconstruction accuracy. In particular, it is very prominent in the feature extraction and dimensionality reduction module. Compared with the classification method based on Bayesian and great likelihood, a large amount of single particle data is required to estimate the relative angle orientation of macromolecular single particles in the 3D structure, and we propose that the clustering method shows good results. CONCLUSIONS: SimcryoCluster can use the contrastive learning method to perform well in the unlabeled high-noise cryo-EM single particle image classification task, making it an important tool for cryo-EM protein structure determination.

Structure-function relationships in ShKT domain peptides: ShKT-Ts1 from the sea anemone Telmatactis stephensoni.

Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (KV 1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit KV 1.x, while others do not. Mutagenesis studies have shown that a Lys-Tyr (KY) dyad plays a key role in KV 1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against KV 1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block KV 1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against KV 1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers.

Advances Toward Amphidinolides C, F and U: Isolations, Synthetic Studies and Total Syntheses.

Amphidinolides C, F, and U, including C2-C4 analogs, are highly cytotoxic marine macrolides, mainly isolated from dinoflagellates of the genus Amphidinium. All these polyketides share a 75 % or more similar structure, highlighted by a macrolactone ring, at least one trans-2,5-substituted-THF motif and a characteristic polyenic side chain. From their isolation and absolute configurational assignment, the total synthesis of these marine macrolides represented an intense challenge to the organic synthesis community over the last 15 years, with around 14 research groups engaged in this inspiring task. In the first part of this review, we present the different approaches to the isolation and characterization of these natural products, including the most recent analogs, which may cast doubt on the biogenetic origin of these compounds. The various synthetic approaches to the total synthesis of C, F, and U amphidinolides are presented in a second part, focusing on key reactions and/or innovative strategies. The review concludes in a third section summarizing the successful approaches leading to the total synthesis of one of the members of this amphidinolide subfamily.

Synthesis, Reactivity, and Complexation with Fe(0) of a Tight-bite Bis(N-heterocyclic silylene).

The synthesis, reactivity, and complexation with Fe(0) precursor of a tight-bite bis(N-heterocyclic silylene) (bis(NHSi)) ligand 1 are reported. The reaction of 1 with p-toluidine led to the activation of both N-H bonds across Si(II) atoms to afford a four-membered heterocyclic cyclodisilazane 2, with hydride substituents attached to five-coordinate Si atoms. A 1 : 2 reaction of 1 with Fe(CO)5 led to an intriguing dinuclear complex 3 featuring a five-membered (N-Si-Fe-Fe-Si) ring with a Fe-Fe bond distance of 2.6892(13) Å. All compounds (1-3) were thoroughly characterized by various spectroscopic methods and X-ray diffraction studies conclusively established their molecular structures. DFT calculations were carried out to shed light on bonding and energetic aspects in 1-3.

Influence of Chemical Modifications of the Crystallophore on Protein Nucleating Properties and Supramolecular Interactions Network.

Crystallophores are lanthanide complexes that have demonstrated outstanding induction of crystallization for various proteins. This article explores the effect of tailored modifications of the crystallophore first generation and their impact on the nucleating properties and protein crystal structures. Through high-throughput crystallization experiments and dataset analysis, we evaluated the effectiveness of these variants, in comparison to the first crystallophore generation G1. In particular, the V1 variant, featuring a propanol pendant arm, demonstrated the ability to produce new crystallization conditions for the proteins tested (hen-egg white lysozyme, proteinase K and thaumatin). Structural analysis performed in the case of hen egg-white lysozyme along with Molecular Dynamics simulations, highlights V1's unique behavior, taking advantage of the flexibility of its propanol arm to explore different protein surfaces and form versatile supramolecular interactions.

'Passivated Precursor' Approach to All-Alkynyl-Protected Gold Nanoclusters and Total Structure Determination of Au130.

A 'passivated precursor' approach is developed for the efficient synthesis and isolation of all-alkynyl-protected gold nanoclusters. Direct reduction of dpa-passivated precursor Au-dpa (Hdpa=2,2'-dipyridylamine) in one-pot under ambient conditions gives a series of clusters including Au22(C≡CR)18 (R=-C6H4-2-F), Au36(C≡CR)24, Au44(C≡CR)28, Au130(C≡CR)50, and Au144(C≡CR)60. These clusters can be well separated via column chromatography. The overall isolation yield of this series of clusters is 40 % (based on gold), which is much improved in comparison with previous approaches. It is notable that the molecular structure of the giant cluster Au130(C≡CR)50 is revealed, which presents important information for understanding the structure of the mysterious Au130 nanoclusters. Theoretical calculations indicated Au130(C≡CR)50 has a smaller HOMO-LUMO gap than Au130(S-C6H4-4-CH3)50. This facile and reliable synthetic approach will greatly accelerate further studies on all-alkynyl-protected gold nanoclusters.

N,N'-Diaryl-Sulfurdiimides are Strongly Redox Tuned.

The synthesis and extensive characterization of nine aryl sulfur diimides (SDIs, Ar-NSN-Ar) are presented with a robust computational and experimental investigation of the fundamental properties of these important members of the thiazyl family of compounds, with particular attention paid to their highly tunable electrochemical behaviour. This is the first work to undertake a systematic comparison of the electrochemical profiles of a coherent series of SDIs to demonstrate and quantify the response of their reduction potentials to substituent electron-donating and -withdrawing properties. This effect is found to be not only exceptionally strong, but also correlates very closely with computed orbital energies. Electron paramagnetic resonance spectroscopy is used to determine the nature, localization, and qualitative lifetimes of the radical anions of SDIs. This work also addresses significant misconceptions about physical properties of SDIs. Experimental data and modern computational methods are employed to provide a resolute answer to the long-standing contention of the solution-state conformations of SDIs, and to correct historical mistakes in the assignment of infrared spectroscopic data. High-quality crystal structures of all SDIs in this work showcase the utility of the recently introduced structural refinement software NoSpherA2, enabling full anisotropic refinement of H-atoms with accurate C-H bond lengths.

Probing the Molecular and Macroscopic Structure of Solid Solutions by Dynamic Nuclear Polarization (DNP) Enhanced 13C and 15N Solid-State NMR Spectroscopy.

Crystallization is a widely used purification technique in the manufacture of active pharmaceutical ingredients (APIs) and precursor molecules. However, when impurities and desired compounds have similar molecular structures, separation by crystallization may become challenging. In such cases, some impurities may form crystalline solid solutions with the desired product during recrystallization. Understanding the molecular structure of these recrystallized solid solutions is crucial to devise methods for effective purification. Unfortunately, there are limited analytical techniques that provide insights into the molecular structure or spatial distribution of impurities that are incorporated within recrystallized products. In this study, we investigated model solid solutions formed by recrystallizing salicylic acid (SA) in the presence of anthranilic acid (AA). These two molecules are known to form crystalline solid solutions due to their similar molecular structures. To overcome challenges associated with the long 1H longitudinal relaxation times (T1(1H)) of SA and AA, we employed dynamic nuclear polarization (DNP) and 15N isotope enrichment to enable solid-state NMR experiments. Results of solid-state NMR experiments and DFT calculations revealed that SA and AA are homogeneously alloyed as a solid solution. Heteronuclear correlation (HETCOR) experiments and plane-wave DFT structural models provide further evidence of the molecular-level interactions between SA and AA. This research provides valuable insights into the molecular structure of recrystallized solid solutions, contributing to the development of effective purification strategies and an understanding of the physicochemical properties of solid solutions.

Isolation and Structure Elucidation of JBIR-157, a Skeletally Novel Aromatic Polyketide Produced by the Heterologous Expression of a Cryptic Gene Cluster.

Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.

Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B-NS3 protease.

Three new series of macrocyclic active site-directed inhibitors of the Zika virus (ZIKV) NS2B-NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3-aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic  d-cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure-based development of improved inhibitors.

CryoEM Workflow Acceleration with Feret Signatures.

Common challenges in cryogenic electron microscopy, such as orientation bias, conformational diversity, and 3D misclassification, complicate single particle analysis and lead to significant resource expenditure. We previously introduced an in silico method using the maximum Feret diameter distribution, the Feret signature, to characterize sample heterogeneity of disc-shaped samples. Here, we expanded the Feret signature methodology to identify preferred orientations of samples containing arbitrary shapes with only about 1000 particles required. This method enables real-time adjustments of data acquisition parameters for optimizing data collection strategies or aiding in decisions to discontinue ineffective imaging sessions. Beyond detecting preferred orientations, the Feret signature approach can serve as an early-warning system for inconsistencies in classification during initial image processing steps, a capability that allows for strategic adjustments in data processing. These features establish the Feret signature as a valuable auxiliary tool in the context of single particle analysis, significantly accelerating the structure determination process.

Host-Guest Cocrystallization of Phenanthrene[2]arene Macrocycles Facilitating Structure Determination of Liquid Organic Molecules.

Single-crystal X-ray diffraction analysis has emerged as the most reliable method for determining the structures of organic molecules. However, numerous analytes, such as liquid organic molecules, pose challenges in crystallization, making their structures directly elusive via X-ray crystallography methods. Herein, we introduced the rapid cocrystallization of a macrocycle named phenanthrene[2]arene (PTA, host) with 15 liquid organic molecules (guests). The guest liquid organic molecules were successively cocrystallized with the aid of the PTA host. Moreover, the chemical structures of the liquid organic molecules could be determined through single-crystal X-ray diffraction analysis. PTA exhibited high adaptivity and was capable of encapsulating liquid organic molecules without forming covalent bonds or strong directional interactions. The results revealed that the adaptive crystals of PTA exhibited excellent cocrystallization capacity. Weak noncovalent interactions between the host and guest molecules were crucial for organizing the guests in an ordered pattern.

Total Synthesis of (+)-Discorhabdin V.

The discorhabdin natural products are a large subset of pyrroloiminoquinone alkaloids with a myriad of biological activities. Despite garnering much synthetic attention, few members have thus far been completed, particularly those featuring a bridging carbon-nitrogen bond that is found in numerous discorhabdins, including discorhabdin V. Herein we report the first total synthesis and full stereochemical assignment of (+)-discorhabdin V. To access the pyrroloiminoquinone we developed a convergent N-alkylation/oxidative aminocyclization/bromination cascade that joins two key components, which are both made on multigram scale. An intramolecular Heck reaction then forms the quaternary carbon center in an intermediate containing the carbon-nitrogen bridge, and a reductive N,O-acetal cyclization sequence introduces the final piperidine ring. Furthermore, we have established the relative configuration of (+)-discorhabdin V through experimental NOESY data and DP4 NMR probability calculations. The absolute configuration of the natural product has also been determined by circular dichroism and the use of an amino acid derived chiral starting material. Our work represents one of only two reports of a total synthesis of a nitrogen-bridged discorhabdin and paves the way for future biological evaluation of such compounds.

Stereochemically Active Tin(II)-Induced Enhancement of Birefringence in SnII SnIV (PO4 )2 and SrSn(PO4 )PO2 (OH)2.

Two new tin(II) phosphates, SnII SnIV (PO4 )2 and SrSn(PO4 )PO2 (OH)2 , were synthesized by the high-temperature solution method and hydrothermal method, respectively. Theoretical study indicates that by introducing tin(II) with stereochemical activity lone pairs (SCALP) in metal phosphates, the birefringence was enhanced, 0.048@1064 nm for SnII SnIV (PO4 )2 and 0.080@1064 nm for SrSn(PO4 )PO2 (OH)2 .

High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy.

Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids-aimed at preserving thin, uniform vitrified ice and improving protein adsorption-have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with up to 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6-Å resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipoproteins by at least 5-fold, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, this approach supports customized grid designs targeting specific proteins, owing to its broad compatibility with a variety of nanomaterials.

Determining the Projected Crystal Structure from Four-dimensional Scanning Transmission Electron Microscopy via the Scattering Matrix.

We present a gradient-descent-based approach to determining the projected electrostatic potential from four-dimensional scanning transmission electron microscopy measurements of a periodic, crystalline material even when dynamical scattering occurs. The method solves for the scattering matrix as an intermediate step, but overcomes the so-called truncation problem that limited previous scattering-matrix-based projected structure determination methods. Gradient descent is made efficient by using analytic expressions for the gradients. Through simulated case studies, we show that iteratively improving the scattering matrix determination can significantly improve the accuracy of the projected structure determination.