Quasicrystals (QCs), a class of aperiodic crystals with long-range order and non-crystallographic rotational symmetry, provide a unique platform for exploring emergent magnetism beyond conventional periodic solids. Although theoretical studies have long suggested that magnetic order is compatible with quasiperiodicity, long-range magnetic order in real QCs has only recently been realized through design principles established from their periodic approximants. This review presents three complementary and hierarchical principles for understanding and controlling magnetism in Tsai-type QCs and periodic approximants: (i) tuning the valence electron concentration (e/a) to control the sign and strength of Ruderman-Kittel-Kasuya-Yosida (RKKY) interactions, (ii) exploiting crystal electric field (CEF) anisotropy to select spin orientation and stabilize noncoplanar magnetic textures, and (iii) controlling structural degrees of freedom to manipulate frustration. Together, these principles provide a phenomenological framework for understanding and organizing magnetic phase selection in quasiperiodic materials. They also provide a unified perspective on the contrasting emergence of long-range magnetic order and spin-glass-like freezing in Tsai-type quasicrystals. Critical behaviour near magnetic transitions, emerging opportunities and outstanding challenges are also discussed, highlighting future directions for discovering novel magnetic states in QCs.
Cryo electron microscopy (cryo-EM) has made great advances in the last decade, progressively increasing its impact in structural biology as a key method to address molecular structures and mechanisms of various macromolecular complexes. Single-particle cryo-EM will soon equal the number of yearly entries in the Protein Data Bank from structures determined by X-ray crystallography. This is largely thanks to improved cryo electron microscope instrumentation and advanced image-processing tools and structure-sorting methods. As the role of cryo-EM is expanding to an increasingly large community, including newcomers and scientists joining from related fields, it is timely to revisit some fundamental concepts and basics of single-particle cryo-EM and image processing as terminology has become less well defined and, in some cases, confusing. Here we summarize and define some typical terms important for understanding the underlying physical concepts. These include `cryo-EM', `cryo-ET', `3D reconstruction', `coarsening', `contour level' and others. We also discuss resolution estimation and map deposition.
Small-angle X-ray scattering (SAXS) provides important information about global biomolecular solution structures yet is resolution limited. Extension to higher scattering angles through wide-angle X-ray scattering (WAXS) extends the resolution of solution measurements to single ångströms; however, interpretation of WAXS profiles is challenging. Here we establish a practical framework for interpreting WAXS measurements in terms of repeated real-space distances and specific structural motifs in nucleic acids. We compare experimentally measured scattering profiles from two 25-base-pair DNA duplexes, an AT-rich construct and a mixed-sequence duplex. Their SAXS profiles are similar but they exhibit strikingly different WAXS signatures. By systematically varying the reciprocal-space cutoff prior to indirect Fourier transforms, we show how increasing information content sharpens real-space resolution and reveals periodic features associated with base stacking and duplex geometry. Real-space distance distributions identify the dominant atomic contributors to these features and directly connect them to backbone separations, groove widths, and long-range stacking periodicities that vary in a sequence-dependent manner. Together, these results establish WAXS as a powerful tool for probing nucleic acid architecture and, in conjunction with time-resolved measurements, dynamics, and demonstrate that WAXS measurements provide a robust and generalizable route to extracting high-resolution structural information from solution measurements.
Free-standing thin liquid films offer a convenient way to probe matter in solution without a substrate, where a small sample thickness is required. The production of thin films in a vacuum environment is required for probes that cannot penetrate through a surrounding gas or in cases where the gas produces problematic background noise. Nozzles that produce continuously flowing liquid sheet jets have been employed recently for scattering and spectroscopy measurements at X-ray free-electron lasers (XFELs). However, their performance in vacuum with aqueous samples is complicated by high freezing susceptibility due to a strong evaporative cooling effect. Here, we introduce a vacuum-compatible gas-impinging liquid sheet jet nozzle capable of generating sub-100 nm aqueous films. The introduction of a second axisymmetric helium gas sheath surrounding the liquid orifice enables seamless startup and shutdown within a vacuum environment. We investigated the sheet geometry dependence under varying flow conditions and liquid viscosity and demonstrated its viability for X-ray scattering measurements at a high-repetition-rate XFEL source. Finally, we present potential strategies for reducing sample consumption. Our findings confirm that this nozzle exhibits simplified vacuum operation and may help promote the broader utilization of liquid sheet jets for several experimental applications.
The structural behaviour of homoleptic xenon difluoride (XeF2) complexes [M(XeF2)6][SbF6]2 (M = Cu, Zn) under varying temperature and pressure has been investigated, aiming to resolve the disordered Jahn–Teller distortions in the copper complex (CuSb). At 200 K, both CuSb and its zinc analogue (ZnSb) crystallize in a layered CdCl2-type structure with the space group R3. Upon cooling below 170 (CuSb) and 160 K (ZnSb), both systems transition to isostructural phases in P1, with CuSb assuming an ordered Jahn–Teller distortion. The transformation is driven by the shortening and optimization of the Xe⋯F intermolecular contacts, forming stronger and more directional interactions, rather than by Jahn–Teller effects alone. This is supported by the observation of similar transitions in the Jahn–Teller-inactive Zn system. High-pressure experiments up to ∼2.8 GPa at room temperature show the structural stability of the high-symmetry phases, implicating kinetic barriers to further transformation. Additionally, the synthesis and structural characterization of a novel arsenic analogue, [Zn(XeF2)6][AsF6]2 (ZnAs), reveal similar layered motifs but distinct phase behaviour. Symmetry-mode analyses relate all observed phases through distortions of a common CdCl2 aristotype.
Large-scale photon and neutron (PaN) experiments generate vast amounts of valuable data, yet implementation of the FAIR principles (findable, accessible, interoperable and reusable) remains incomplete. Metadata catalogs are a key tool for addressing this challenge. They do not store data itself but provide structured searchable descriptions that make datasets discoverable and reusable. This article, developed within the DAPHNE4NFDI consortium of Germany's National Research Data Infrastructure (NFDI), reflects the discussion on the role of metadata catalogs in PaN science such as SciCat, ICAT and SampleDB from a scientific point of view, and presents the status of current implementations. Integration of standards such as NeXus, sample databases and electronic laboratory notebooks is also addressed. Catalog implementations are categorized by purpose as raw data, institutional data and public data, and examined by practical aspects including authentication and authorization, persistent identifiers, and metadata enrichment. Drawing on case studies such as the RefXAS X-ray absorption spectroscopy database, we summarize lessons learned and present recommendations for minimizing fragmentation, enabling interoperability, and harmonizing identity and persistent-identifier policies. Looking ahead, semantic technologies and knowledge graphs will play a central role in building a federated FAIR-compliant catalog infrastructure that supports both facility and university researchers.
Single-particle cryoEM has made visualization of biological molecules and their complexes at high resolution possible without the need for crystallization. The last decade has seen a rapid growth in the number of structures determined by cryoEM; for membrane proteins, the total number of cryoEM structures has exceeded that determined by crystallography.
Ultrafast pump-probe serial femtosecond crystallography (SFX) experiments are typically performed with extremely high excitation laser fluence to maximize the occupancy of the light-induced state. This has been justified by assuming that a dominant fraction (up to 99%) of the pump laser light is scattered by the jetting medium, strongly reducing the pump laser fluence in the crystals which, therefore, do not absorb multiple photons and thus undergo the biologically relevant single-photon photoreaction. However, this notion is strongly contested in the field. To address the issue of pump laser scattering losses in high viscosity jets, we performed time-resolved SFX experiments on fatty acid photodecarboxylase crystals using high-viscosity extrusion (HVE) and both patterned and non-patterned sheet-on-sheet (SOS) fixed-target sample delivery systems on the Cristallina-MX instrument at SwissFEL. Tightly focused pump laser beams were used to mitigate light contamination (the undesired accidental illumination of neighboring crystals) in SOS chips, paving the way for their use in pump-probe investigations of lipidic-cubic-phase-grown membrane protein crystals. Determination of the light-induced reaction intermediate occupancies enabled a comparison of the influence of the delivery method on the pump laser fluence reaching the crystals. We show that optically transparent high-viscosity jets and chip crystal delivery methods result in photoproduct yields that approach 70-80% and 60-70%, respectively, of computationally derived yields that take into account excitation probabilities. These results demonstrate that clear viscous jets scatter pump laser light far less than has been claimed. Consequently, the use of excessive photoexcitation energy densities is neither needed nor justified in optically pumped time-resolved SFX experiments when using appropriately sized crystals.
Structural phase transitions in metastable rutile-related V 0.92 O 2 synthesized at 10 GPa and 1273 K were studied with single-crystal X-ray diffraction in the temperature range 110–500 K and at pressures up to 9.2 GPa. V 0.92 O 2 starts to decompose at 470 K and atmospheric pressure. When heated to temperatures above about 350 K, the material transforms to an incommensurate phase. The oxygen sublattice is essentially rigid and it is mostly the V atoms that are affected by the modulation. An anharmonic description of the displacement parameters and their corresponding modulation is used for the V atoms to reach satisfactory agreement factors for main and first-order satellite reflections, indicating substantial disorder in the modulated structure. Measurements of electronic transport properties provide evidence that the incommensurate phase is insulating. On compression at room temperature, V 0.92 O 2 reaches the ideal rutile structure at about 5.0 GPa. Both structural and electronic phase transitions are of the first-order character. The results of this work demonstrate that the structural and electronic behaviour of V 0.92 O 2 at extreme conditions is distinctly different from that of stoichiometric VO 2 .
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.
The entry of SARS-CoV-2 into cells is mediated by a trimeric spike protein that utilizes its receptor-binding domain (RBD) to engage with the cell surface receptor ACE2, with each protomer capable of shuttling between an `up' conformation that can bind ACE2 and a `down' conformation that prevents ACE2 binding. To discover intermediate conformational states during this transition, we applied and refined a recently reported linear subspace method to embed 2D projection cryo-EM images of ACE2-bound spike proteins into a low-dimensional latent space. From this embedding, multiple conformational states were reconstructed followed by an automated masking protocol, leveraging a topology representing network and optimal transport. A surprising result from our analysis is the finding that the dominant trajectory for the transition between conformations that have one-up and two-up RBD states involves an all-down RBD conformational state. The methods we present here could be generally applicable to other dynamic protein assemblies to uncover novel intermediate conformational states.
Recent progress in gas dynamic virtual nozzle (GDVN) technologies in combination with high-brilliance synchrotron and X-ray free-electron lasers (XFELs) has allowed the visualization of protein dynamics in crystallo by mixing macromolecular protein crystals with a substrate using tunable mixing times on the order of milliseconds to seconds prior to serial X-ray diffraction data collection. This has become the method of choice for high-resolution structure determination of intermediate states. However, such experiments require large counts of crystals of proper sizes for high-resolution data collection, and premium beam times for screening efforts. Cryogenic microcrystal electron diffraction (MicroED) represents a complementary technique that may be a more accessible avenue for time-resolved nanocrystallography compared with serial X-ray diffraction experiments. MicroED can produce full diffraction datasets from just a few submicrometre-thick crystals, and the approach is more readily accessible, requiring standard cryogenic transmission electron microscopy (TEM) equipment available at many universities and institutes. Cryogenic MicroED, like other forms of cryo-EM, begins with rapidly freezing biological material on electron microscopy grids. In the case of MicroED, micro- to nano-crystals (<500 nm thick) are deposited onto electron microscopy grids and plunge-frozen for subsequent electron diffraction data collection. Here, we have incorporated GDVN technology developed originally for XFEL experiments into the freezing process as a first step towards time-resolved studies. We describe the limited deposition efficiency of the model MicroED protein proteinase K on TEM grids using GDVNs, preceding sample vitrification and successful MicroED data collection. We discuss both the initial results from such experiments and the methodological challenges in developing this approach into a reliable workflow for millisecond-to-second time-resolved structural studies of macromolecules. Our results promise a strategy to deposit crystals on grids using GDVNs and determine high-resolution structures by MicroED, constituting a first step towards development of time-resolved MicroED experiments.
We review the structural and functional characteristics of bacteriophages and bacteriocins (diffocins) that specifically target Clostridioides difficile , a significant healthcare concern due to its role in nosocomial infections. The advent of modern cryogenic electron microscopy (cryoEM) has revolutionized our understanding of these contractile injection systems, providing high-resolution insights into their mechanisms. We compare the structures of C. difficile phages and diffocins, highlighting their adaptations for penetrating the Gram-positive bacterial cell envelope – including the cell membrane, cell wall and proteinaceous surface layer. Diffocins, simpler in structure, utilize a combination of mechanical and enzymatic strategies, while some phages like ΦCD508 may rely on mechanical force alone. This review delves into the assembly and function of key components such as the contractile sheath, baseplate and receptor-binding proteins, offering a framework for engineering precision antimicrobials. We also present new experimental results, including refined cryoEM structures of the ΦCD508 pre- and post-contracted tail, a novel spontaneously contracted conformation and an X-ray crystal structure of a phage receptor-binding protein domain. This work underscores the potential of cryoEM in advancing our understanding of phage biology and its applications in developing targeted therapies against C. difficile .
S-Adenosyl methionine (SAMe), the biological methyl donor essential for sustaining the life of most complex organisms, is the second most widely used cofactor, after ATP, in biochemical reactions and is synthesized by the enzyme methionine adenosyl transferase (MAT) from ATP and methionine. MAT, also known as S-adenosylmethionine synthetase, is found in almost every organism. SAMe is employed universally by different methyltransferases that catalyze the methylation of biomolecules such as nucleic acids, proteins and lipids. In plant cells SAMe produced by MAT enzymes controls the level of critical metabolites such as ethylene, polyamines and biotin, and regulates essential cellular processes such as cell division and synthesis of cell wall, chlorophyll and membrane. MAT enzyme complex MATα2β, comprising the catalytic unit MATα2 and the regulatory protein MATβ, is found in nearly all human tissues and is essential for providing the necessary SAMe flux for methylation of DNA and various proteins including histones. The enzymatic activity of MATα2 is enhanced by several fold upon complexation with both variants of MATβ (βV1 and βV2). Using cryogenic electron microscopy, we determined the high-resolution resting-state structures of the MATα24βV12 and MATα24βV22 complexes, providing insights into the allosteric regulation of MAT catalytic activity, revealing how MATβV association could facilitate substrate binding, stabilize the transition state and promote product release to drive the catalytic cycle, and opening new possibilities for inhibitor binding.
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.
Copper-containing nitrite reductases (CuNiRs) catalyse the reduction of nitrite to nitric oxide and are a key enzyme in the anaerobic ammonium oxidation and denitrification steps of the nitrogen cycle. The recent recognition of the widespread distribution of three-domain CuNiRs where cognate redox partners are fused to the core NiR enzyme offered the possibility of studying coordinated events ( e.g. proton-coupled electron transfer) in a conformationally stable donor–acceptor complex. The C-terminal cytochrome c tethered domain of the CuNiR from Ralstonia pickettii ( Rp NiR) has been well studied. Reverse engineering of Rp NiR undertaken to remove the cognate partner domain showed that the presence of the additional domain resulted in significant differences in the apparent K m for nitrite and the reduction potentials of the Cu centres when compared with the core enzyme. The oxidation state of the haem centre and the position of the tethering linker have also been shown to control access of substrate to the active site. A key feature of this control is a conserved tyrosine residue (Tyr323 in Rp NiR) located in the tethering linker between the fused domain and the core enzyme. To gain insight into this control, we have undertaken targeted mutations of Rp NiR to probe the so-called primary proton channel and perturb putative electron transfer routes from the haem to the `gatekeeper' Tyr323 and to the T1Cu centre. The resolution of our crystallographic data to better than 1.2 Å enabled us to apply unrestrained SHELXL refinement of the structures. Our data provide a significant advance in our understanding of catalysis and modulation of electron transfer in these tethered systems, with wider implications for these fundamental processes in other protein complexes.
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.
We introduce a compact mathematical formulation for the inverse single-particle 3D reconstruction problem, a high-dimensional inverse problem in which millions of parameters are estimated from extremely noisy experimental measurements. Given a collection of noisy 2D projection images (particles) of an unknown 3D charge-density distribution, the objective is to infer the unknown particle orientations and thereby enable ab initio 3D reconstruction via tomographic methods. We develop a method for generating regularized reprojections directly from the noisy particles that does not rely on explicit 3D density reconstruction. Instead, we recast the ab initio orientation-recovery problem in polar Fourier coordinates through discretization of the rotation group SO(3). The directions of projection are mapped onto slices intersecting the origin of the 3D Fourier transform. The rotations in the plane normal to a projection direction are mapped onto radial lines in the 2D Fourier transforms of the particles. An optimization procedure jointly estimates particle projection directions, in-plane rotation angles and rotational origin offsets. Regularized reprojections are computed by averaging along lines in the polar Fourier representation, exploiting data redundancy to suppress noise and improve stability. We present the mathematical framework in detail and provide initial benchmarks demonstrating the performance and robustness of the approach.
High-temperature polymorphs of hafnia (HfO 2 ) are of significant interest in electronics and fuel-cell applications, and stabilization at ambient conditions can be achieved by aliovalent substitution and nanosize effects. Y 3+ 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 Hf 1– x Y x O 2– 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% Y 3+ . The average structure conforms to the cubic fluorite phase of HfO 2 , 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 Zr 3 Y 4 O 12 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.
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.
