Screening for novel chemical scaffolds targeting PCNA identifies the Hsp90alpha inhibitor SNX-2112.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the main human enzyme that deposits the most abundant internal mRNA modification, N6-methyladenosine (m6A). In the heterodimeric complex, METTL3 acts as a catalytic subunit while METTL14 is involved in mRNA binding and complex stabilization. Here, we present the discovery of small-molecule ligands that bind to a cryptic pocket in METTL14 by protein crystallography. A comparative analysis of crystal structures revealed that the METTL14 cryptic pocket is closed in the apo structure of METTL3-14, and in the structures of METTL3-14 in the complex with the cosubstrate S-adenosyl-methionine (SAM) and a large number of SAM-competitive inhibitors. We first discovered compounds 1 and 2 that bind to both the SAM pocket in METTL3 and the cryptic pocket in METTL14. With this structural information, we designed compound 3 that binds only to the METTL14 cryptic pocket. Compound 3 does not inhibit the catalytic activity of METTL3-14 but can be used as an anchor for heterobifunctional molecules. We propose a route for its further development into heterobifunctional ligands, e.g., proteolysis targeting chimeras (PROTACs).

The replacement of aromatic rings with saturated molecular frameworks is a recent development encapsulated by the motto "escape from flatland" and conceptualized through the use of saturated benzene bioisosteres. This Review summarizes the application of the smallest bicyclic and spirocyclic ring systems as saturated scaffolds, focusing on applications in constructing bioactive molecules. We discuss considerations of their molecular strains, their potential to serve as saturated benzene isosteres in terms of both volume and geometry, and structural data derived from small-molecule and protein crystallography. Additionally, we present general approaches to synthesis, examine the current commercial availability of functional building blocks, and present existing examples of applications of bicyclic systems in drug discovery programs. At least eight structures based on the smallest skeletons have advanced to clinical trials, with one, vanzacaftor, recently receiving U.S. FDA approval. Our analysis indicates that small bicyclic fragments are represented exceptionally unevenly. While bicyclo[1.1.1]pentane and spiro[3.3]heptane have become routine and indispensable in medicinal chemistry over the past decade, ladderane and housane remain exotic and unexplored. We highlight knowledge gaps, aiming to stimulate interest in small saturated skeletons for innovative molecular engineering.

Mycobacterium tuberculosis isocitrate lyase 2 (ICL2) is an allosterically regulated enzyme required for growth on non-glycolytic carbon substrates during infection. Although acetyl-CoA and its analogues are known to activate ICL2, the molecular basis of this regulation has remained unclear. Here, we combine protein NMR, crystallography, molecular dynamics, and mutagenesis to show that two structural features unique to ICL2, the C-terminal domain and a helical substructure in the N-terminal catalytic domain, govern its allostery. Acetyl-CoA binding promotes dimerisation of the C-terminal domain and disrupts its contacts with the helical substructure to trigger conformational changes that activate the enzyme. Together, these findings reveal how a non-substrate metabolite drives isocitrate lyase activation, uncovering the allosteric mechanism that controls M. tuberculosis metabolism and informs new therapeutic strategies.

Viruses are numerically the most abundant forms on Earth, and most are present in soil. Even though viruses are highly abundant in soil and critical to rhizosphere function, visualizing the diverse morphotypes within soil has been challenging. The difficulty is primarily due to the heterogenous nature of isolated suspensions that typically contain nanometer to micron scale debris which renders protein crystallography for structural studies unfeasible and hinders cryo-electron microscopy due to ice thickness and contrast issues. Here we employed and compared a simple spin filtration method to cleanup solutions of extracted viruses for direct observation with cryo-electron microscopy. The method employs common physical biochemical separation steps to remove large and small debris which dramatically improves image quality and preservation of structural features to permit visualizing morphotypes not typically seen with conventional negative stain approaches. In addition to tailed and non-tailed polyhedral phages, several under reported or novel morphotypes of soil viruses are directly visualized as a particle library with both 2D and 3D information.

This perspective essay recounts fundamental aspects of two forms of racemic protein crystallography, techniques that significantly enhance the success rate for determining protein molecular structures by X-ray diffraction. Crystallization from a racemic mixture of protein enantiomers, i.e., the natural chirality L-protein and its D-protein enantiomer, gives highly ordered centrosymmetric crystals with a success rate much greater than for the L-protein alone and even facilitates the crystallization of L-protein molecules proven to be recalcitrant to crystallization by conventional methods. X-ray reflections from such centrosymmetric racemic protein crystals have quantized phases, greatly simplifying solution of protein structures by direct methods and giving high-quality electron density maps. Quasi-racemic mixtures of protein isomorphs (proteins with mirror image shapes that are not true chemical enantiomers) also strongly facilitate the formation of diffraction-quality crystals. D-protein molecules are prepared by total synthesis based on modern chemical ligation methods. Examples selected from the literature will be highlighted to illustrate the application and utility of racemic crystallography for the elucidation of protein structures.

We present the development of a piezo-actuated shutter system designed for high-power broadband X-ray beams at fourth-generation synchrotron sources. The device combines a flexure-based mechanical design with efficient water cooling, achieving full open-close transitions in 2 ms while reliably withstanding continuous thermal loads exceeding 20 W. This performance enables safe operation with multilayer monochromators, which introduce higher heat loads than conventional optics such as Si(111) monochromators. The shutter provides an open aperture of 1.9 mm, is fully ultrahigh vacuum compatible, and was validated through mechanical and thermal characterization under realistic operating conditions. Its millisecond-scale actuation allows precise blocking of the beam during idle phases such as sample alignment or repositioning, thereby minimizing radiation damage and maximizing the effective use of the delivered photon flux. This makes the system particularly well suited for high-throughput scanning and imaging techniques, including ptychography, tomography, scanning small-angle X-ray scattering and protein crystallography, on modern synchrotron beamlines.

X-ray crystallography remains a powerful technique for determining high-resolution protein structures; however, obtaining high-quality crystals is a significant bottleneck. This study presents a detailed experimental workflow that employs differential scanning fluorimetry (DSF) to optimize protein crystallization. DSF, which measures protein thermal stability, was used to refine both protein buffer composition and crystallization conditions. The method was applied to two distinct proteins: CreD, a nitrosuccinate lyase, and HIRA, a histone chaperone. For CreD, DSF-based optimization of the protein buffer enhanced the crystal quality, increasing the resolution from 3.32 to 2.18 Å. For HIRA(644-1017), DSF-guided optimization of the protein buffer significantly improved the protein solubility from 0.1 to 19.1 mg ml-1, facilitating the growth of initial crystals. Further optimization of the crystallization conditions using DSF, combined with microseeding, improved the crystal quality, leading to structure determination at 2.45 Å resolution. This study demonstrates that DSF is a valuable tool for efficiently optimizing protein crystallization. The workflow presented here, involving initial DSF-based optimization of protein buffers followed by DSF-guided optimization of crystallization conditions, offers a rational approach to enhancing protein crystal quality, thereby facilitating structure determination by X-ray crystallography.

Abstract Introduction: Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the United States and the second leading cause of cancer related deaths. A new therapeutic strategy that can treat a large population of patients is necessary. One emerging direction is cancer metabolism; however, targeting metabolic pathways poses a toxicity risk due to these pathways being active in both benign and malignant cells. Prior studies demonstrate that more advanced stages of PCa are more dependent on glycolysis. We have developed a compound, SGI-1553, which selectively inhibits the early steps in glycolysis in prostate cancer cells and patient-derived xenografts (PDXs), but does not affect benign cells at therapeutic levels. However, the exact target of this compound was not known. The purpose of this study was to identify the target of SGI-1553. Methods: Lysates from LNCaP and LNCaP95 cells were treated with SGI-1553 or DMSO. Lysates were then exposed to increasing temperatures, digested, and run through mass spectrometry. Proteins showing a thermal shift in the denaturing temperature when exposed to SGI-1553 were identified. Protein crystallography of the lead target and compound were then performed. Enzyme assays of the recombinant target protein exposed to increasing doses of SGI-1553 were run. The target was knocked down in LNCaP and LNCaP95 cells using a shRNA lentiviral construct. RNA-seq on control and knockdown cells was then performed followed by GSEA pathway analysis. Results: Thermal shift mass spectrometry revealed that the target of SGI-1553 is adenosine kinase (ADK). Treatment with SGI-1553 resulted in a 5˚C thermal shift to the right for ADK when compared with DMSO. Crystallography of recombinant ADK with SGI-1553 demonstrated binding of SGI-1553 within the adenosine pocket of ADK. Enzyme assays of recombinant ADK showed increased activity of ADK above baseline with increasing concentrations of SGI-1553 (15-18% max increase). In shADK cells compared with control cells, GSEA analysis demonstrated upregulation of Myc targets-V1 and V2, E2F targets, and fatty acid metabolism; downregulation was seen in hypoxia and epithelial to mesenchymal transition pathways. These same pathways were up and downregulated in human PCa xenografts that were resistant to the effects of SGI-1553. Conclusion: SGI-1553 binds to and activates ADK in PCa cells that are responsive to the inhibitory growth effects of SGI-1553. shADK cells displayed similar regulation of pathways on GSEA analysis as human PDXs that are unresponsive to SGI-1553 in vivo. Adenosine kinase has numerous roles in cells including energy regulation, nucleotide pool regulation, and regulation of transmethylation. Our group is currently examining the effects of overexpressing and knocking down ADK expression in prostate cancer cell lines on glycolysis, proliferation, and methylation. Identifying ADK as the target and examining its biological effects in prostate cancer will allow us to use structure-guided drug development to create more potent compounds for use in prostate cancer therapy. Citation Format: Shihua Sun, Ilsa Coleman, Lyssa Weible, Kathryn Soriano Epilepsia, Mika Munari, Peter Nelson, Robert Moritz, Scott Lovell, Stephen Plymate, Wesley Van Voorhis, Kayode K. Ojo, Cynthia Sprenger. A novel selective glycolysis inhibitor, SGI-1553, targets adenosine kinase in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr B072.

Prospects for neutron protein crystallography at the European Spallation Source.

We provide a historical introduction spanning the past 50 years of synchrotron radiation protein crystallography. We then provide a resume of current trends. These help us to celebrate the huge influence that synchrotron radiation, and now X-ray lasers, has had on the scope of protein crystallography. It has also accelerated the development of closely allied methods such as neutron protein crystallography, which has adopted the synchrotron Laue method as its own aswell as developing monochromatic and time-of-flight methods. Also, the democratic access to central synchrotron facility beamlines has prompted similarly operated centres of electron cryo-microscopy and micro-electron diffraction. We offer our thoughts on the current trends across this scientific landscape.

Protein crystallography is a key technique of structural biology that allows researchers to determine the three-dimensional structure of proteins, protein-protein, protein-ligand, and protein-nucleic acid complexes at the atomic level. Whereas obtaining high-quality crystals is the first milestone for the structural characterization of a protein, the establishment of a detailed crystallization protocol promotes reproducibility of the crystallization process. Here, we describe a versatile crystallization method for human D-dopachrome tautomerase (D-DT or MIF-2), a small pleotropic protein of increasing interest due to its key role in human pathophysiology. The protocol described here provides a step-by-step procedure for generating high-quality protein crystals for a wide range of D-DT variants, including aggressive truncations and mutants with mechanistic value.

Hirshfeld atom refinement (HAR) provides a more realistic interpretation of crystallographic data than the standard independent atom model (IAM) by using aspherical atomic form factors derived from quantum mechanical (QM) calculations. With this aspherical description, it is possible to obtain improved atomic positions, atomic displacement parameters and correct bond lengths even for hydrogen atoms. Unfortunately, HAR is computationally very demanding for larger molecules. Recently, we suggested how this can be solved by calculating aspherical atomic form factors for small overlapping fragments of the system, the fragHAR approach. Here, we have created a new implementation of fragHAR in Olex2 within the NoSpherA2 interface. We have also solved previous issues with hydrogen bonds by automatically extending the fragments with all hydrogen-bond acceptors. This implementation was successfully tested on three oligopeptides, demonstrating that fragHAR yields indistinguishable results in terms of atomic charge, residual density or R values compared with full HAR. Subsequently, fragHAR was applied to the proteins crambin and rubredoxin, with 843 and 1014 atoms, respectively, showing improved results in terms of egross, which decreases from 0.350 with the IAM to 0.318 with fragHAR for crambin, and from 0.195 to 0.176 for rubredoxin, although it turned out to be necessary to keep all bond lengths involving hydrogen atoms constrained for the latter protein. FragHAR shows near-linear scaling and 46-fold speedup for rubredoxin compared with HAR. It also provides a convenient solution to alternative conformations and positional disorder, which cause an exponential increase in the time consumption of the conventional HAR approach. The successful refinement of rubredoxin marks a significant milestone, presenting the first HAR application of a metalloprotein, and further underlines the relevance of fragHAR in protein crystallography.

The curious life of human mitochondrial SOD2.

X-ray crystallography has long been the workhorse technique for enabling the analysis and investigation of 3D protein structures. This understanding is crucial for deciphering protein function, including enzymatic reactions, signaling pathways, and more. The initial step in this process involves the crystallization of the target protein. In this pursuit, we have developed a microfluidic device that leverages an electrically-actuated strategy for fluid handling, built on a LEGO®-inspired architecture. This device enables on-demand control of counter-diffusive mixing by decoupling reagent loading from mixing, harnessing surface forces without necessitating pumping connections. The LEGO®-based architecture involves gold-LEGO®-electrodes (GLEs) that are snug fit into a device fabricated by photolithography and nanoimprinting. Our approach entails straightforward pipetting of crystallization reagents into the device to set up counter-diffusion crystallization, followed by the application of <1 V to trigger fluid mixing, thus creating a 'valve' that can be easily actuated using AAA batteries, all encompassed into a 150 μm thin device. Fabrication of the device using an X-ray transparent polymer allows for in situ X-ray crystallography, obviating the need for subsequent extraction and mounting of the protein crystals, and streamlining the process of protein structure determination. Using our LEGO®-based electrically-actuated protein crystallization and X-ray crystallography (LEAP-X) platform, we have successfully demonstrated the utility of the device using lysozyme, thaumatin, and proteinase K as model proteins, as well as the crystallization and in situ, room temperature structural analysis of the metalloprotein rubrerythrin as a novel target. Lastly, we propose the utility of this platform for the addition of chemical triggers for time-resolved protein crystallography.

Three-dimensional electron diffraction（3D ED）, also known as microcrystal electron diffraction（MicroED）, has emerged as a powerful technique for determining the structures of ultra-thin protein crystals. Originally developed for protein crystallography, 3D ED/MicroED was subsequently demonstrated to be applicable to small-molecule crystallography for the micrometer-scale crystals. In this manuscript, we describe the development of a semi-automated 3D ED/MicroED data collection and processing system at the University of Tsukuba and its operation as a shared-use facility.

Advances in structural biology increasingly focus on uncovering protein dynamics and transient macromolecular complexes. Such studies require modeling of low-occupancy species like time-evolving intermediates and bound ligands. In protein crystallography, difference maps that compare paired perturbed and reference datasets are a powerful way to identify and aid modeling of low-occupancy species. Current methods to generate difference maps, however, rely on manually tuned parameters and, when signals are weak due to low occupancy, can fail to extract clear, chemically interpretable signals. We address these issues, first by showing that negentropy – a measure of how different a signal looks from anticipated Gaussian noise – is an effective metric to assess difference map quality and can therefore be used to automatically determine difference map calculation parameters. Leveraging this, we apply total variation denoising, an image restoration technique that requires a choice of regularization parameter, to crystallographic difference maps. We show that total variation denoising improves map signal-to-noise and enables us to estimate the latent phase contribution of low-occupancy states. We implement this technology in an open-source Python package, METEOR. METEOR opens new possibilities, for time-resolved and ligand-screening crystallography especially, allowing detection of low-occupancy states that could not previously be resolved.

Biosynthesis of hydroxy terpenes, which possess better solubility and target-binding capability than terpene hydrocarbons, generally needs cascaded catalysis of terpene synthase (TS) and cytochrome P450 oxygenase. Interestingly, some TSs can directly generate hydroxy terpenes (mostly monohydroxy terpenes) independent of oxygenases. There are even rare TSs that can form dihydroxy terpenes directly. Nevertheless, the structure and catalytic mechanism of dihydroxy terpene synthases (DHTSs) remain elusive to date, hindering their practical applications. Through protein crystallography, multiscale simulations, and site-directed mutagenesis, we elucidate a stepwise carbocation quenching mechanism. In this process, two water molecules are strictly manipulated by a dynamic hydrogen-bond network to quench the carbocation intermediates. Most importantly, Tyr312 was identified as the indispensable and irreplaceable residue for initiating the reprotonation of the monohydroxy terpene. The spatiotemporally precise manipulation mechanism of DHTSs enriches the knowledge of TSs and lays a foundation for developing an oxygenase-independent biosynthesis system of multihydroxy terpenes.

LigPCDS (Ligand Point Cloud Data Set) is the first dataset of chemically labeled 3D point clouds of protein ligands. 3D images and structures of ligands were derived from X-ray protein crystallography experimental datasets deposited at the Protein Data Bank. The 3D point cloud format allowed for a computer-comprehensive representation of the ligand’s experimental data, enabling the interpretation of the ligand’s chemical structure using a building block-like labeling approach. For constructing LigPCDS, the images of the ligands were interpolated from their difference electron density map into a 3D grid-like structure, filtered around their atomic spheres, and stored in point clouds. The density value was used as a single feature. Chemical vocabularies, based on atoms and their cyclic structural arrangements, were designed and used to pointwise label these 3D representations of the ligands. The proposed imaging and labeling approaches were validated by training semantic segmentation deep learning models on a stratified dataset from LigPCDS, which could recover the protein ligand’s chemical structure with good performance. LigPCDS can be used to achieve solutions for building known and yet unknown protein ligands (small organic molecules) from experimental X-ray protein crystallography, in silico ligand screening, drug design, and to understand protein function in basic biology.

Photorhabdus bacteria live in mutualistic relationships with Heterorhabditis nematodes, and together, they act as effective insect pathogens. These bacteria produce a diverse array of lectins, sugar-binding proteins that are believed to play crucial roles in the complex tripartite interaction among Photorhabdus, nematodes, and their insect hosts. One such lectin, Photorhabdus laumondii tumor necrosis factor (TNF)-like lectin (PLTL), identified in Photorhabdus laumondii subsp. laumondii TTO1, exhibits notable sequence similarity to the N-terminal domain of the BC2L-C lectin (BC2L-CN), a TNF-like lectin recognized for its specificity toward fucosylated glycans associated with human embryonic stem cells and certain cancers. Through glycan array analysis and surface plasmon resonance, we identified PLTL's binding preference for branched histo-blood group oligosaccharides. The crystallographic structure of PLTL in complex with the BLeb pentasaccharide reveals a network of direct and water-mediated hydrogen bonds simultaneously stabilizing the Fucα1-2 and Galα1-3 moieties, which define its narrow glycan specificity. A combination of mass spectrometry, protein crystallography, and analytical ultracentrifugation showed a unique hexameric PLTL architecture stabilized by intermolecular disulfide bridges. Our data suggest that PLTL may contribute to the mutualistic relationship between Photorhabdus and its nematode symbiont, Heterorhabditis bacteriophora, rather than playing a role in the interaction with the insect host. This study provides a structural and functional characterization of PLTL, a newly identified member of the TNF-like lectin family. Comparative analysis with BC2L-CN highlights both conserved and distinct structural features, suggesting potential applications in glycan recognition-based diagnostics or biotechnological tools beyond its biological role. Our findings underscore its complex glycan specificity and offer insights into its potential role in Photorhabdus-nematode symbiosis.