Search Results (422)

Small gases pose an unusual problem for studies of molecular transport in proteins. O₂, CO, H₂, and NO can cross short-lived internal spaces opened by protein fluctuations, often faster than experiments can follow continuous migration. Time-resolved crystallography can localize sufficiently populated intermediates, whereas spectroscopy, isotope exchange, and kinetic measurements report molecular exchange over their respective timescales without resolving the complete route. Pressurized noble-gas structures expose internal accommodation sites but rely on surrogate molecules whose size and interactions differ from those of physiological gases. Geometry-based tunnel searches identify available space, while molecular dynamics follows explicit movement through a fluctuating protein. Free-energy and enhanced-sampling approaches can access states or transitions that remain undersampled in direct trajectories. These techniques resolve different quantities rather than progressively more accurate estimates of gas transport. In this Perspective, we argue that gas-migration pathways should be evaluated by the physical consistency of independent observables, with each method interpreted according to the quantity it resolves. This distinction explains why a cavity visible crystallographically may not carry substantial flux, why a rapidly crossed route can remain structurally inconspicuous, and why static narrowing can alter diffusion without predicting its magnitude. Agreement among methods can support a transport assignment when the quantities they resolve are physically consistent with the same mechanism; apparent disagreement may instead reflect differences among occupancy, accessibility, residence, energetic preference, and molecular traffic. Full article

Precise site-specific ligand design remains a major challenge in structure-based drug discovery. Most existing approaches screen ligands against binding pockets identified from static protein structures obtained by X-ray crystallography, NMR spectroscopy, cryo-electron microscopy, or AlphaFold predictions. However, protein function is governed by a structure–dynamics–function relationship, and ligand screening that does not account for binding competition across the protein surface or the receptor’s dynamic, substrate-dependent conformational states remains incomplete. Substrate-specific conformations are underexplored and may offer new opportunities for selective ligand design, although systematic workflows to identify and exploit such states remain limited. Previously, we showed that collagen alters matrix metalloprotease-1 (MMP1) dynamics and that R405 is a collagen-specific allosteric residue exhibiting strong dynamic correlations with the catalytic site. Here, we present a computational framework for substrate-specific allosteric ligand design using collagen-bound MMP1 as a model system. We characterized the conformational dynamics of free and collagen-bound MMP1 by all-atom molecular dynamics simulations, clustered the resulting conformational ensembles, and identified conformations unique to the collagen-bound state. These conformations were used as structural templates for machine-learning-based generation of approximately 150,000 candidate ligands, which were subsequently docked against both the R405-centered region and all detectable binding pockets on the MMP1 surface. Several candidate ligands were predicted to dock preferentially at the R405 region by at least 0.3 kcal/mol compared with competing surface pockets. Together, these results establish a generalizable computational workflow for identifying candidate ligands predicted to preferentially dock to substrate-specific allosteric conformations and provide a foundation for future experimental validation of selective allosteric modulation. Full article

The authors previously reported that 2-methylbenzimidazole derivatives (MBIs) exhibit insect growth-regulating activity against the silkworm, Bombyx mori. However, despite their unique effects on juvenile hormone (JH)-related endocrine pathways, the precise mode of action of MBIs remained unclear. In the present study, the interactions between MBIs and the lepidopteran hemolymph JH-binding protein (JHBP), a key regulator of JH transport and activity, were investigated using multiple approaches, including in vitro binding affinity assays, X-ray crystallography, and molecular docking simulations. A series of MBIs bearing a 1-(4-alkoxybenzyl) group, which exhibited potent insect growth-regulating activity, showed high binding affinity and structural compatibility with the JH-binding pocket of JHBP. In contrast, 1-(4-alkylbenzyl) MBIs, which displayed weak or negligible insect growth-regulating activity, exhibited low affinity for JHBP. These findings suggest that the insect growth-regulating activity of MBIs is mediated through inhibition of JHBP function, likely by disrupting the precise regulation of JH concentration in the hemolymph during larval development and pupal metamorphosis. Full article

Monoclonal antibodies (mAbs) have been the subject of extensive study in recent years due to their recognition as highly promising therapeutic molecules offering high specificity and a low risk of side effects. Monitoring the structure of these molecules is crucial for developing new therapeutics, characterizing interactions with antigens or receptors, and explaining potential changes in activity between antibody production batches. However, commonly used biophysical approaches provide only low-spatial-resolution information, and conventional structural biology techniques such as crystallography and cryo-electron microscopy (cryo-EM) are difficult to apply to these highly dynamic proteins. Solution nuclear magnetic resonance (NMR) spectroscopy is the method of choice for structural studies of flexible proteins at atomic resolution; however, it has traditionally been limited to low-molecular-weight biological systems. In this review, we present recent advances in NMR spectroscopy and advanced isotopic labeling methods that have enabled the atomic-resolution study of both the crystallizable (Fc) and antigen-binding (Fab) fragments of antibodies. We show how NMR is becoming a powerful tool for investigating full-length mAbs at an atomic level, opening up new possibilities for the characterization and in-depth quality control of therapeutic antibodies in solution. Full article

The photoreceptor bacteriorhodopsin (HsBR) from Halobacterium salinarum is a model system for studying ultrafast photoinduced reactions in proteins. Recent time-resolved serial femtosecond crystallography (TR-SFX) experiments require high pump energies, raising concerns about nonlinear excitation and multi-photon effects. Here, we systematically investigate the influence of excitation energy, pulse duration and the sign of the chirp on the initial HsBR photo-reaction using femtosecond Vis-pump IR-probe spectroscopy in the retinal C=C stretching region. An acousto-optic programmable dispersive filter enabled independent control of pulse energy and chirp. Within the tested range, the retinal dynamics were independent of pulse duration and chirp, indicating that fluence alone does not fully describe excitation conditions. Increasing excitation energy leads to nonlinear saturation of the retinal signals and the appearance of an additional band near 1550 ${\mathrm{cm}}^{-1}$. However, this band rises linearly with the excitation energy. Hence, the additional band is not directly caused by non-resonant multi-photon absorption. Spectral decomposition reveals two components: a low-energy contribution consistent with the known retinal isomerization dynamics and a high-energy contribution attributed to a small population of photo-damaged HsBR likely formed via a resonant two-photon process. These findings clarify the role of excitation conditions in ultrafast HsBR spectroscopy and suggest that spectral changes at high pump energies mainly arise from damaged species upon resonant two-photon excitation. Full article

X-ray crystallography is still the most widely used and versatile method for structural studies of biological macromolecules. This study concerns the application of nanogels to facilitate protein crystallization, a prerequisite for X-ray crystallography. Nanogels (NGs) are nano-sized, highly crosslinked polymeric particles that have been extensively studied for chemical catalysis and drug delivery but not for protein crystal nucleation. The efficacy of six types of nanogels (three N-isopropylacrylamide-based and three acrylamide-based) was tested, with promising results. They were subsequently functionalised with active hydroxyl groups for further testing. Both functionalised and non-functionalised nanogels were tested on model (trypsin, thaumatin, proteinase K, ferritin and catalase) and target proteins (glulisine, α-crustacyanin and acriflavine resistance protein subunit AcrB) using both manual and automated techniques. All nanogels were found to be effective in promoting protein crystallization in both screening and optimization trials, giving crystal ‘hits’ that would have otherwise been missed. Overall, the functionalised nanogels were more effective. Nanogel effects are proposed to be due to a combination of surface porosity and surface chemistry. Full article

Psychrophilic organisms are able to grow at temperatures down to −15 °C, while hyperthermophiles can multiply at temperatures up to 122 °C. What structural changes in extremophile proteins are needed to maintain stable and biochemically active structures under such conditions? Understanding how such extremophiles accomplish this is relevant for human health, biotechnology, and our search for life elsewhere in the universe. The purpose of the current study is to report and compare the structures of four rubredoxins (Rds), the first ever two experimental psychrophile bacteria structures (from Gram-positive Clostridium psychrophilum and Gram-negative Polaromonas glacialis) and two hyperthermophiles from the Gram-negative Thermotoga maritima bacterium and the archaeon Pyrococcus yayanosii, also a piezophile, as part of a program to understand structural variations that support both stability and function under extreme conditions. These structures were obtained using synchrotron radiation X-ray diffraction at 100 K. All four structures had the expected overall rubredoxin fold. Rubredoxin from the only aerobic psychrophilic bacterium Polaromonas glacialis had larger variations in sequence and structure, whereas the other psychrophilic bacterium showed properties closely related to hyperthermophile rubredoxins. Multi-subunit structures showed similar RMSD variability independent from their thermal adaptation status. We propose including functional information in the analysis since temperature optimization may not be the only determinant for a specific protein adaptation. Full article

X-ray free-electron lasers (XFELs) have revolutionized structural biology by enabling “diffraction-before-destruction” and capturing the ultrafast dynamics of life. However, the intrinsic sparsity and noise of XFEL diffraction snapshots, often complicated by multi-lattice overlaps, create a formidable computational bottleneck that limits data utilization and structural fidelity. Here, we present MCDPS-SFX, a robust indexing framework based on a reference-based, whole-pattern matching principle integrated with parallelized iterative refinement. By exhaustively sampling orientation space and progressively rejecting outliers, MCDPS-SFX significantly outperforms legacy algorithms—more than doubling crystal yields in heterogeneous datasets (e.g., 21,807 vs. 8792 for MOSFLM)—and achieves highly competitive yields comparable to state-of-the-art indexers, such as extracting over 90,000 lattices in the lysozyme benchmark. We demonstrate its efficacy on standard benchmarks and technically demanding G-protein-coupled receptor (GPCR) systems, including the rhodopsin–arrestin complex and the glucagon receptor. MCDPS-SFX consistently produces high-quality data statistics, enabling the high-resolution visualization of functionally critical, flexible regions such as phosphorylated receptor tails. Our results provide a powerful tool for enhancing the scientific output of XFEL experiments, offering a robust alternative for maximizing information recovery from weakly diffracting or overlapping crystalline samples. Full article

Background: Point mutations to the androgen receptor (AR) ligand-binding domain (LBD) are becoming increasingly recognized as a mechanism of therapeutic resistance in castration resistant prostate cancer (CRPC). The present review explores how point mutations induce molecular changes that contribute to the eventual treatment failure of androgen receptor pathway inhibitors (ARPIs) in CRPC. Methods: The PubMed database was searched for structural studies on the AR LBD. Eligible articles included molecular docking analysis and emphasized changes in ligand–receptor interactions after point mutation. Structural data were obtained from the Protein Data Bank (PDB) using the search parameters “Androgen receptor ligand binding domain”, “Homo sapiens”, and “X-ray diffraction”. PDB files of wild-type and point mutant AR LBDs were accumulated for analysis. Results: A functional shift from inhibiting to activating AR has been documented for multiple ARPIs. Crystallography data and in silico evaluation have deciphered how changes in steric hindrance of the AF-2 domain contribute to ARPI loss of function. To combat therapeutic resistance, discovery efforts have begun to consider combination approaches of orthosteric and allosteric inhibitors, as well as compounds that target other AR domains. Although lead compounds have been identified, none have progressed into the clinic. Conclusions: Questions remain regarding the best approach for rationally designing new AR targeting therapeutics. Understanding how structural changes to the AR LBD lead to the failure of clinical therapeutics is a necessary step that should precede drug discovery campaigns. Moreover, computational modeling is a powerful tool that should be leveraged to streamline therapeutic development. Full article

Protein crystals are intrinsically hydrated systems, and their structural integrity is strongly influenced by environmental humidity. Understanding the effects of relative humidity (RH) variation on crystal stability is therefore essential for both fundamental research and applied studies. In this work, the structural response of tetragonal hen egg-white lysozyme (HEWL) to controlled RH variation was investigated using in situ X-ray powder diffraction (XRPD). Polycrystalline HEWL samples were subjected to systematic gradual dehydration and rehydration cycles, as well as to non-gradual RH variation protocols. Pawley analysis of the XRPD data enabled monitoring of the evolution of unit cell parameters and unit cell volume as a function of RH. Under all experimental conditions, the tetragonal polymorph (space group P4₃2₁2; a = 79.105 (4) Å, c = 38.231 (2) Å) was preserved. RH variation induced smooth, continuous and anisotropic lattice changes, characterized by a decrease in the a (=b)-axis and a concomitant increase in the c-axis upon dehydration, while rehydration resulted in the opposite behavior. The overall magnitude of lattice variation remained limited (within ±2%), indicating a high degree of structural stability. Partial degradation of crystallinity was observed only after prolonged exposure to low RH levels. These findings demonstrate the remarkable structural resilience of tetragonal HEWL and highlight the effectiveness of in situ XRPD as a powerful tool for probing hydration-driven lattice responses in protein crystals under realistic environmental conditions. Full article

Background/Objectives: Follitropin delta is an approved recombinant follicle-stimulating hormone (rFSH) expressed in a human cell line. Correct disulfide connectivity is a critical quality attribute for rFSH, a heterodimeric glycoprotein composed of noncovalently associated α and β subunits and stabilized by an extensive network of intramolecular disulfide bonds. Disulfide characterization is typically performed by mass spectrometry (MS). However, the closely spaced disulfide bonds within the FSH α-subunit are particularly resistant to proteolytic cleavage, complicating conventional MS-based disulfide mapping. Methods: To overcome limitations of MS-based methods, an X-ray crystallography strategy was employed using a ternary complex of the recombinant FSH heterodimer with an anti-FSHα Fab and a stabilizing anti-kappa VHH. Crystals of the desialylated rFSH/Fab/VHH complex were obtained and diffraction data were collected. Results: The structure of recombinant FSH was determined at 2.29 Å resolution. Electron density surrounding cysteine residues in both the α and β subunits was well defined, allowing unambiguous assignment of all intramolecular disulfide bonds in the crystallized protein. The observed cysteine connectivity is fully consistent with the disulfide architecture of FSH from other sources and supports correct folding of the recombinant Follitropin delta. Full article

G-quadruplexes (G4s) are specialized nucleic acid structures extensively formed throughout the genome, with particular enrichment in regulatory regions such as telomeres, promoters, and transcriptional enhancers. These four-stranded assemblies are involved in multiple chromosomal processes, including DNA replication, transcription, maintenance of genomic stability, and epigenetic regulation, and are closely associated with cancer biology. Due to their unusual thermodynamic stability, G4s serve as physical barriers to DNA/RNA unwinding, thereby impeding replication, transcription, and translation and compromising genome integrity. To mitigate this threat, cells have evolved dedicated helicases that can actively resolve G4 structures. In this review, we summarize recent structural advances—primarily derived from protein crystallography—regarding the mechanisms by which helicases unwind G4 quadruplexes. The insights presented herein establish a framework for elucidating the molecular basis of G4 unfolding and for the rational design of small-molecule G4 ligands and therapeutic agents. Additionally, we explore the applications of G4 helicases in nanopore sequencing, which aim to enhance sequencing accuracy, throughput, and continuity. Full article

The broadly neutralizing monoclonal antibody 3D1 potently neutralizes SADS-CoV by targeting a conserved epitope within the heptad repeat 1 (HR1) domain of the viral spike protein. Structural and biophysical analyses demonstrate that 3D1 binds with high affinity to a specific linear β-turn motif (residues A804–N809) in HR1. High-resolution crystallography reveals that this motif sits within a deep, electrostatically complementary paratope groove. Critically, 3D1 binding competitively inhibits the essential interaction between HR1 and HR2. Notably, its recognition is not dependent on HR1’s native helical conformation, as it maintains strong binding to conformationally constrained, stapled helical peptides. Collectively, the data indicate that 3D1 neutralizes by capturing a pre-hairpin intermediate state of HR1—a transition state between prefusion and postfusion forms—thereby sterically blocking the formation of the stable postfusion six-helix bundle that is essential for membrane fusion. This work defines a precise, structure-dependent neutralizing epitope and elucidates a mechanism of action that involves trapping a key fusion intermediate, offering a valuable template for the design of broad-spectrum coronavirus therapeutics. Full article

Direct methods provide a model-free approach to solving the crystallographic phase problem and deliver unbiased atomic structures. However, conventional iterative projection algorithms such as Hybrid Input–Output (HIO) face two critical challenges: discontinuous density modification at the protein-solvent boundary and inaccurate molecular envelope reconstruction that fails to account for trapped solvent, particularly in crystals with solvent content approaching the lower limits of direct phasing applicability. We introduced four continuous iterative projection algorithms, including our improved continuous version, which implements smooth density modification at protein-solvent interfaces. To address envelope inaccuracy, we developed a two-step refined reconstruction scheme using sequential large-radius and small-radius Gaussian filters to identify trapped solvent molecules within surface cavities and internal channels. This scheme enhances the performance of both continuous and classical algorithms, including HIO, the difference map, and our improved versions. Benchmarking on 28 protein structures (solvent contents 55–78%, resolutions 1.46–3.2 Å, reported R-factor less than 0.22) showed that the refined envelope scheme increased average success rates of continuous algorithms by 45.7% and classical algorithms by 60.5%. The performance of continuous algorithms and improved classical algorithms proved comparable to the well-established HIO algorithm, forming a top-tier group that exceeded other classical algorithms. Integrating a genetic algorithm co-evolution strategy further enhanced average success rates by approximately 2.5-fold and accelerated convergence through population-wide information sharing. Although the success rate correlates with solvent content, our strategy improved success probability at any given solvent level, extending the practical boundaries of direct methods. The high success rate enabled averaging of multiple independent solutions, which reduced mean phase error by approximately 6.83° and yielded atomic models with backbone root-mean-square deviation (RMSD) typically below 0.5 Å relative to structures reported in the Protein Data Bank (PDB). This work introduces novel algorithms, a refined envelope reconstruction methodology, and an effective optimization strategy with genetic algorithm evolution. The complete framework enhances the capability and reliability of direct methods for phasing protein crystals with limited solvent content and provides a toolkit for addressing challenging cases in structural biology. Full article

Direct methods for solving protein crystal structures from X-ray diffraction data provide an essential approach for validating predicted models while avoiding external model bias. Nevertheless, traditional iterative projection algorithms, including the widely used Difference Map (DiffMap), are often limited by modest phase retrieval success rates. To address this limitation, we introduce a novel Hybrid Difference Map (HDM) algorithm that synergistically combines the strengths of DiffMap and the Hybrid Input–Output (HIO) method through six distinct iterative update rules. HDM retains an optimized DiffMap-style relaxation term for fine-grained density modulation in protein regions while adopting HIO’s efficient negative feedback mechanism for enforcing the solvent flatness constraint. Using the transmembrane photosynthetic reaction center 2uxj as a test case, the first HDM formula, HDM-f1, successfully recovered an atomic-resolution structure directly from random phases under a conventional full-resolution phasing scheme, demonstrating the robust phasing capability of the approach. Systematic evaluation across 22 protein crystal structures (resolution 1.5–3.0 Å, solvent content ≥ 60%) revealed that all six HDM variants outperformed DiffMap, achieving 1.8–3.5× higher success rates (average 2.8×), performing on par with or exceeding HIO under a conventional phasing scheme. Further performance gains were achieved by integrating HDM with advanced strategies: resolution weighting and a genetic algorithm-based evolutionary scheme. The genetic evolution strategy boosted the success rate to nearly 100%, halved the median number of iterations required for convergence, and reduced the final phase error to approximately 35° on average across test structures through averaging of multiple solutions. The resulting electron density maps were of high interpretability, enabling automated model building that produced structures with a backbone RMSD of less than 0.5 Å when compared to their PDB-deposited counterparts. Collectively, the HDM algorithm suite offers a robust, efficient, and adaptable framework for direct phasing, particularly for challenging cases where conventional methods struggle. Our implementation supports all space groups providing an accessible tool for the broader structural biology community. Full article