The paper by Gilski et al. in this issue [IUCrJ (2026), 13, 132-145] focuses on the stereochemical analysis of the catalytic mechanism of asparaginases with the aim of resolving key ambiguities in the mechanism. These enzymes have found truly remarkable therapeutic success in the treatment of childhood leukemias.
The systematic comparison of 3D-ED methods in terms of data quality presented by Schmitt et al. [IUCrJ (2026), 13, 291-303] is discussed.
Support by National Science Centre Poland under grant No. UMO-2023/51/B/ST5/02843 for the work described in the article by Grabowski et al. [(2025), IUCrJ, 12, 403-416] is acknowledged.
The near-universal adoption of electron cryo-microscopy (cryo-EM) by structural and cell biologists has led to exponential growth of the field, especially over the last two decades, with a doubling in the number of deposited electron-microscopy density maps every 2.5 years. This exponential growth has changed in recent years to become linear. Cryo-EM methods are now able to contribute to our structural understanding of biological complexity ranging from atomic resolution maps of assemblies of multiprotein complexes to in situ investigation of protein structures in the context of intact cells, snapshots of multiple conformations and insights into fundamental chemical mechanisms underlying biological function. Combined with dramatic advances in artificial intelligence, the increasing democratization and implementation of cryo-EM appears poised to drive a new revolution in digital biology.
Ideal symmetry is known to break down under almost any noise. One measure of asymmetry in a periodic crystal is the relative multiplicity Z' of geometrically non-equivalent units. However, Z' discontinuously changes under almost any displacement of atoms, which can arbitrarily scale up a primitive cell. This discontinuity was recently resolved by a hierarchy of invariant descriptors that continuously change under all small perturbations. We introduce a Continuous Invariant-based Asymmetry (CIA) to quantify (in physically meaningful ångstroms) the deviation of a periodic crystal from a higher-symmetry form. Our experiments on several crystal structure prediction datasets show that about a half of simulated crystals have high values of CIA, while all experimental structures in these datasets have CIA = 0. On another hand, many crystals with high values of Z' in the Cambridge Structural Database (CSD) turned out to be close to more symmetric forms with Z' ≤ 1 due to low values of CIAs.
High-temperature polymorphs of hafnia (HfO2) are of significant interest in electronics and fuel-cell applications, and stabilization at ambient conditions can be achieved by aliovalent substitution and nanosize effects. Y3+ stabilization of hafnia (YSH) introduces local cation disorder around charge-compensating oxygen-ion vacancies, and here we establish both the average and local structure of YSH nanoparticles using synchrotron powder X-ray diffraction (PXRD) and pair distribution function (PDF) analysis. A range of phase-pure crystalline nanoparticles of Hf1-xYxO2-x/2 were prepared via continuous flow solvothermal synthesis and subsequent high-temperature annealing, and full stabilization of the cubic phase is achieved already at 13 at% Y3+. The average structure conforms to the cubic fluorite phase of HfO2, but local displacive disorder caused by electrostatic attraction of neighbouring oxygen ions and repulsion of neighbouring metal ions by the net-positive oxygen-ion vacancies is established. The well-known Zr3Y4O12 structure, which incorporates such relaxation motifs, provides a good proxy description of YSH. In situ X-ray total scattering experiments provide insight into the formation mechanism of the YSH nanoparticles and initial precipitation of an atomically mixed amorphous phase is followed by crystallization over several minutes. The crystallization rate increases with higher reaction temperature, whereas an increased doping level results in slower crystallization.
We report the cryo-EM structure-guided discovery of GND-135, a novel small-molecule inhibitor of the VCP/p97 AAA ATPase that demonstrates efficient inhibition of VCP/p97 in biochemical, cellular, and pharmacokinetic assays and in a tumor efficacy mouse model of acute myeloid leukemia. Our approach overcomes the liability in the clinical-stage compound CB-5083 where Phase I studies showed off-target activity of CB-5083 for the enzyme PDE6. From the cryo-EM structural analysis of CB-5083 bound to PDE6 and VCP/p97, we identified critical ligand/protein interactions in both proteins and rationally designed a small molecule that retains key interactions necessary for VCP/p97 inhibition while eliminating PDE6 off-target activity. We refer to this approach as `subtractive optimization' because we are leveraging our ability to determine both on-target and off-target cryo-EM structures to guide the medicinal chemistry campaign to enable more targeted compound design. While this strategy is not possible in all cases, the use of cryo-EM to tune on-site binding while eliminating off-target binding could be a generally applicable strategy for informing molecular design and accelerating small-molecule drug discovery.
Advances in serial crystallography have reshaped approaches to radiation damage at room temperature. This commentary highlights recent work comparing XFEL and synchrotron experiments, emphasizing its implications for serial crystallography at modern X-ray sources.
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.
Combining powder X-ray and neutron diffraction, the first crystallographic characterization of diacetylene (IUPAC designation 1,3-butadiyne) is presented and supplemented by Raman spectroscopy and periodic-DFT calculations. The structure is described in the space group Pnma, with a = 9.348 (2), b = 5.9890 (6), c = 5.6746 (11) Å and V = 317.69 (6) Å3 at 5 K, with four molecules in the unit cell resulting in a density of 1.0466 g cm-3. The diacetylene molecules are arranged in a layered structure dominated by C-H...π interactions, which leads to anisotropic thermal expansion behaviour. No structural phase transitions were observed across the 5-220 K temperature range at ambient pressure. The structural similarity with acetylene identifies it as a potential cocrystal component of particular importance for Titan's surface chemistry in view of the upcoming NASA Dragonfly mission.
The m = 2 member of the monophosphate tungsten bronze family had been thought to be the only one without an electronic instability at low temperature. In this paper, we report the discovery of a charge density wave phase in this compound, with a transition temperature of 290 K and an incommensurate modulation vector q = 0.245b* + ξc*, which reaches a lock-in with a commensurate vector at 130 K. The presence of this new phase is confirmed by diffraction and resistivity measurements. Pre-transitional dynamics are investigated using diffuse and inelastic X-ray scattering, revealing a clear Kohn anomaly. We analyze both structural and electronic contributions to the phase transition, providing a comprehensive picture of the mechanism driving this newly identified instability.
In this issue of IUCrJ, Subramaniam, Kühlbrandt & Henderson present an overview of the remarkable progress that has been made in electron cryo-microscopy and electron cryo-tomography.
A correctly solved crystal structure should agree with the experimental data, and its geometry should correspond to a local minimum on the potential energy surface (PES). The idea of verifying crystal structure solutions by comparing them with their geometry-optimized versions was introduced 15 years ago. Recent developments in machine learning interatomic potentials (MLIPs) have made it possible to replace computationally expensive density functional theory (DFT) calculations with AI/neural-network-based alternatives. MLIPs can reach DFT-comparable precision with a substantial gain in speed. We selected one promising MLIP, Universal Models for Atoms, trained on the Open Molecular Crystals 2025 dataset, and processed a prefiltered subset of 216 919 structures from the Cambridge Structural Database. Due to the limitations of the MLIP available when this study commenced, ionic compounds, salts and metal-containing structures were excluded. The current methodology cannot process disordered structures, and available computational resources limit the maximum unit-cell volume that can be treated to 4000 Å3. All structures in the dataset were geometry optimized using the MLIP, and similarity descriptors were calculated to quantify the differences between the original and optimized structures. Automatic analysis was followed by the manual identification of issues indicated by the descriptors' values. We detected anomalies in experimental structures that had already passed all prior validation, as well as limitations in the reliability of the MLIP PES calculations. For 1867 crystal structures, bond-pattern change was observed, while 3331 structures showed a root-mean-square Cartesian displacement greater than 0.25 Å. Future improvements to the methodology and extension to systems not covered by this study are discussed.
Interest in electron diffraction (ED) for structural characterization of both proteins and small molecules has grown significantly over the last decade. While ab initio phasing methods remain the gold standard for ED data from small-molecule samples, radiation beam damage during data collection and poor crystallinity of the nanocrystalline sample can make this method unfeasible - particularly for challenging molecules that exhibit conformational flexibility. Molecular replacement (MR) is the most commonly used phasing method for protein ED data and can circumnavigate issues related to diminished data quality. However, its application to small molecules has been limited due to the lack of methods for generating optimal trial conformations. Herein, a high-throughput automated molecular replacement workflow has been developed to solve a novel ED structure of corilagin, a macrocyclic gallotannin with pharmaceutical relevance, which could not be solved with ab initio phasing. The method was validated against three similar macrocycles with known structures (paritaprevir-α, paritaprevir-β and grazoprevir) at varying data resolution limits (1.0, 1.2, 1.4, 1.5, 1.6, 1.8 and 2.0 Å). At all these resolutions for all three structures, the developed workflow was successful and produced solutions with R factors and RMSD values within acceptable limits of the ab initio solved structure.
Obtaining high-quality single crystals containing charged molecules from aqueous solutions can sometimes be challenging. In this publication, we studied the crystallization of polycationic porphyrins. Crystals of three polycationic porphyrins, [5,10,15,20-tetrakis(1-methylpyridin-1-ium-4-yl)porphyrin, 5-(4-cyanophenyl)-10,15,20-tris(1-methylpyridin-1-ium-4-yl)porphyrin and 5-(4-carboxyphenyl)-10,15,20-tris(1-methylpyridin-1-ium-4-yl)porphyrin], were obtained with different anions. We started initially with `nano-crystallization' screening in order to identify promising combinations of the cations with suitable anions, followed by optimizing the ratio of anions to cations. Both steps yielded crystals, leading to the determination of one porphyrin-containing crystal structure from each step. In the next step, five different organic solvents were trialled as additives in an aqueous hanging-drop crystallization setup, which led to the determination of three more crystal structures. It was shown that it was possible to improve the quality of the crystals without the organic additive being incorporated into the crystal structure. Finally, crystals of one porphyrin salt were obtained by gel crystallization. These crystals were higher quality than the crystal grown by vapour diffusion, leading to a high-quality and higher-resolution X-ray structure determination. This is the first porphyrin structure to be determined from a crystal grown by gel crystallizaton.
In the era of single-particle cryogenic electron microscopy (cryo-EM) and AI-driven protein structure prediction, obtaining high-resolution protein structures, either experimentally or computationally, has become increasingly routine. Yet studying and understanding protein dynamics remains challenging. In single-particle cryo-EM, protein dynamics are most obviously manifested as poor local resolution or disappearing densities in specific regions of a reconstruction. No method is yet available to computationally generate conformational ensembles that fully deconvolute these experimental observations. When dynamics are key to understanding protein function, it is clear to us that introducing new experimental approaches is necessary to close this gap and make sense of invisible densities in single-particle cryo-EM.
CryoEM reconstructions must be performed along the Ewald spheres to achieve resolutions beyond the projection approximation limit. The linear theory of image formation models the scattering from the specimen and focusing by the objective lens as two conjugate Ewald spheres that correspond to the two halves of the contrast transfer function (CTF). A focal series of micrographs mimics the focal gradient inherent in the Ewald wavefront, and its 3D Fourier transform provides a visual representation of the conjugate Ewald spheres. To better understand the nature of the Ewald spheres, focal series of graphene oxide as a thin specimen and platinum-iridium as a strongly scattering specimen were acquired. An algorithm was developed to fit and correct the CTF of the whole focal series. Correcting for the one half of the CTF flattens one of the Ewald spheres in the 3D transform, while doubling the curvature of the other. This yields the best representation of the Ewald sphere and thus the `exit wave', and can be performed on every single micrograph. The 2D reconstruction as the sum of the partial CTF corrected transforms recovers missing information in the individual micrographs and increases the signal-to-noise ratio. Iterative refinement of the reconstructed image against the amplitudes of the original focal series recovers the second Ewald sphere only at frequencies where the two spheres overlap, but introduces artifacts. The conclusion is that the correct way to account for the Ewald spheres in cryoEM is to apply the two halves of the CTF to a particle image and incorporate the two results into their corresponding spheres within the cryoEM reconstruction.
We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40-90 pl; ∼40-55 µm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 µm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.
The dynamic nature of protein and macromolecular complexes means that the capture of multiple sequential states along a reaction pathway can provide much greater insight into function than that obtained from a single static structure. We present a set of modular, easy-to-implement tools and workflows for optical excitation, on-grid characterization and tightly coupled rapid vitrification, establishing a proof-of-principle framework for time-resolved cryoEM and cryo-electron tomography (cryoET). We apply this framework to E. coli chemotaxis, in which serine-sensitive chemoreceptors initiate signalling upon ligand binding and undergo critical conformational changes within the chemosensory arrays. Using DMNB-caged serine [O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine] as a model trigger, we quantified its photophysical properties and uncaging efficiency using UV-Vis spectroscopy and two-dimensional gas chromatography mass spectrometry (GC×GC-MS). Coupling a femtosecond-pulsed laser to a Vitrobot enabled reproducible reaction-to-vitrification delays of ∼150 ms, yielding intact E. coli minicells with well-preserved chemotaxis arrays suitable for in situ structural analysis by cryoET. This integrated approach provides a robust and generalisable framework for millisecond time-resolved cryoET, laying the groundwork for capturing transient conformational states in their native cellular context.