Template matching (TM) is a long-standing tool in cryo-electron tomography (cryo-ET), where it has been applied to localize macromolecular complexes across a wide range of sample types. Recent algorithmic and computation...Template matching (TM) is a long-standing tool in cryo-electron tomography (cryo-ET), where it has been applied to localize macromolecular complexes across a wide range of sample types. Recent algorithmic and computational advances have substantially increased the speed, accuracy, and practical resolution of the technique, enabling TM with fine angular sampling and voxel sizes that allow extraction of structural information beyond particle detection and toward exploration of higher-order spatial organization. In addition, deep learning (DL) approaches have emerged as powerful alternatives for particle picking and segmentation. Together, TM and DL form complementary components of modern cryo-ET workflows. This review discusses recent methodological advances in both approaches and highlights how they are increasingly integrated into cutting-edge pipelines for tomography exploration.
Electron ptychography is an advanced four-dimensional scanning transmission electron microscopy technique that demonstrates significant advantages in revealing ultra-high-resolution atomic structures through computationa...Electron ptychography is an advanced four-dimensional scanning transmission electron microscopy technique that demonstrates significant advantages in revealing ultra-high-resolution atomic structures through computational phase retrieval in materials science while offering potential benefits for radiation-sensitive biological specimens. Recent advancements in hybrid pixel array detectors and computational algorithms have enabled low-dose imaging, demonstrating the feasibility of imaging frozen-hydrated biological samples, termed cryo-electron ptychography. Nevertheless, challenges such as low-dose constraints and complexities in computational and experimental setups currently limit the widespread adoption of cryo-electron ptychography and achievable resolution beyond the subnanometer level. This review examines the progress in cryo-electron ptychography, identifies the challenges that distinguish biological specimens from radiation-tolerant samples, and assesses potential future developments in algorithms and hardware that could unlock its capabilities for structural analysis of biomacromolecules and in situ subcellular structures.
Chromatin architecture - encompassing epigenetic states and three-dimensional (3D) nuclear organization - operates at the interface of genotype, cellular metabolism, and transcriptional regulation. Chromatin loops physic...Chromatin architecture - encompassing epigenetic states and three-dimensional (3D) nuclear organization - operates at the interface of genotype, cellular metabolism, and transcriptional regulation. Chromatin loops physically connect gene promoters with distal regulatory elements, providing a structural basis through which non-coding genetic variants modulate target gene expression and contribute to metabolic disease risk. Although the precise mechanisms linking loop dynamics to transcriptional output remain incompletely understood, 3D genomic approaches are rapidly advancing our ability to interpret non-coding variation in disease. Beyond genetic predisposition, chromatin architecture responds dynamically to environmental signals, including diet and circadian oscillations, in a cell-type-specific manner. Here, we review recent advances in mammalian 3D chromatin biology and their implications for transcriptional regulation in metabolic health and disease.
Describing conformational changes in biomolecules using molecular dynamics simulations requires defining an appropriate low-dimensional mathematical description of the system, referred to as a set of collective variables...Describing conformational changes in biomolecules using molecular dynamics simulations requires defining an appropriate low-dimensional mathematical description of the system, referred to as a set of collective variables (CVs). No single CV design strategy is universally optimal; the choice should be guided by the biological question, the property of interest, the evaluation criterion, and the chosen sampling method. Here, we discuss the physical principles that should inform CV design and categorize existing approaches. We also evaluate the relationship between different types of CVs, the amount of data required to train them, and suitable enhanced sampling approaches. Finally, we outline practical guidelines for selecting CVs, helping practitioners match methodological choices to the underlying dynamical process and to the goals of their simulations.
Water-soluble polymers are widely used as model crowders, yet their effects on proteins are often interpreted using frameworks developed for rigid spherical depletants. Here we review polymer crowding from the perspectiv...Water-soluble polymers are widely used as model crowders, yet their effects on proteins are often interpreted using frameworks developed for rigid spherical depletants. Here we review polymer crowding from the perspective of scaling theory, emphasizing how polymer-specific length scales govern protein-polymer interactions across concentration regimes. In dilute solutions, depletion is set by the polymer radius of gyration and scales linearly with concentration. Above the overlap concentration, c∗, the relevant length becomes the correlation length, ξ(c), which defines the mesh size and controls both the magnitude and range of interactions. Protein association, folding, and intrinsically disordered protein structure follow distinct scaling regimes determined by the ratio of protein size to ξ. Deviations from classical predictions arise from polymer connectivity and soft protein-polymer interactions. The polymer-scaling perspective provides a unified framework linking polymer physics to protein thermodynamics in crowded environments.
Biomolecular condensates form through liquid-liquid phase-separation of multivalent biomolecules, enabling the spatiotemporal control of biochemical processes within the cellular environment. They can selectively partiti...Biomolecular condensates form through liquid-liquid phase-separation of multivalent biomolecules, enabling the spatiotemporal control of biochemical processes within the cellular environment. They can selectively partition different components, maintain ionic gradients, and form/dissolve spontaneously in feedback loops, all without the need for a membrane or energy expenditure. Within the cell, the composition, size, localisation and rheology can tune these biomolecular condensates to perform specific biological functions; however, how this happens is not fully understood. Rationally designed biomolecular condensates provide a "bottom-up approach" towards gaining molecular-level insights into what makes condensates optimal for their biological roles. In this review, we discuss how engineered protein condensates outline emerging relationships between design parameters and physicochemical tuneability, and how these systems may be adapted for biological applications.
Intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles, making them particularly sensitive to the crowded intracellular environment. Defining how molecular crowding reshapes these ensemb...Intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles, making them particularly sensitive to the crowded intracellular environment. Defining how molecular crowding reshapes these ensembles is therefore essential for bridging in vitro biophysical observations with cellular function. Recent studies have shown that crowding does not simply drive non-specific compaction. Instead, it remodels IDP conformational ensembles through competing entropic, enthalpic and solvent-mediated contributions, giving rise to diverse and sequence-dependent outcomes. In this review, we summarize recent experimental and computational advances that reveal how distinct classes of crowders modulate IDP conformations and how these effects are further tuned in cellular environments. We also discuss the consequences of crowding-induced ensemble remodeling for molecular recognition, biomolecular phase separation, and aggregation. Together, these findings establish molecular crowding as a key determinant of IDP conformational landscapes and functional behavior in complex biological settings.
Cryogenic electron microscopy (cryo-EM) has transformed structural biology by enabling near-atomic resolution imaging of macromolecules and the direct visualization of molecular architectures in native cellular environme...Cryogenic electron microscopy (cryo-EM) has transformed structural biology by enabling near-atomic resolution imaging of macromolecules and the direct visualization of molecular architectures in native cellular environments. However, conventional cryo-EM, although outstanding at elucidating molecular density and morphology, provides little direct information about chemical composition or molecular state. Here, we review recent advances in scanning-based and spectroscopic electron microscopy that extend cryo-EM beyond phase contrast, including Z-contrast imaging, energy-dispersive X-ray spectroscopy, and electron energy-loss spectroscopy. We highlight how these techniques enable label-free mapping of elemental distributions and chemical states in beam-sensitive biological specimens, and we discuss emerging workflows that integrate spectroscopy with cryogenic sample preparation and correlative imaging. Together, these developments position spectroscopic cryo-EM as a powerful approach for linking structure, composition, and function across molecular and cellular scales.
Early enthusiasm for the "cellular revolution" in cryo-electron tomography (cryo-ET) was largely driven by the promise of resolving protein structures in native environments. However, a parallel trajectory has emerged th...Early enthusiasm for the "cellular revolution" in cryo-electron tomography (cryo-ET) was largely driven by the promise of resolving protein structures in native environments. However, a parallel trajectory has emerged that centers less on structure determination and instead on quantitative information about the nanoscale organization of organelle membranes and associated macromolecules. Together, these advances have shifted the field from descriptive visualization toward conceptual insight, and in the process, redefined what "local" means in organelle biology. In this review, I highlight recent studies that show how cryo-ET has illuminated specialized organelle membrane states defined by geometry, bilayer properties, protein patterning, and molecular sociology. These innovations reveal insights into membrane microenvironments and further establish cryo-ET as a bridge between structural and cell biology.
This review focuses on how cryo-electron tomography (cryo-ET) is contributing to the structural cell biology of eukaryotic chromatin in situ (inside cells) and on the technologies needed to unite the structural cell biol...This review focuses on how cryo-electron tomography (cryo-ET) is contributing to the structural cell biology of eukaryotic chromatin in situ (inside cells) and on the technologies needed to unite the structural cell biology and 3-D genomics fields. Cryo-ET is a form of cryo-EM in which a frozen-hydrated sample is imaged from multiple views; these 2-D images are then computationally processed into a 3-D image called a cryotomogram. Cryo-ET is applicable to purified/constituted nucleosome arrays, isolated chromosomes, isolated nuclei, cells, and tissues. The imaging of cells and tissues makes in situ cryo-ET a powerful approach to study chromatin structure inside cells in a life-like state. This field is in its infancy and lacks the throughput and scalability of genomics. Once several key labeling and image-analysis technologies mature, "structural genomics" will provide both nanometer 3D spatial resolution and base-pair sequence-space resolution to address fundamental mechanisms of gene regulation inside cells.
Understanding how proteins function requires a description of their structural dynamics. Accessing these structural dynamics across time and length scales calls for different methods and often their combination. Here we...Understanding how proteins function requires a description of their structural dynamics. Accessing these structural dynamics across time and length scales calls for different methods and often their combination. Here we review a combination of two highly synergistic and complementary methods-single-molecule Förster resonance energy transfer (smFRET) and hydrogen-deuterium exchange mass spectrometry (HDX-MS). We describe how they complement each other in the ability to resolve structural and dynamical heterogeneity, coverage of protein residues, and types of protein motions they probe. Together, they access a wide range of protein dynamics from global rigid body motions to local unfolding. This synergy has led to the elucidation of activation and regulatory mechanisms in signaling and transcription, which we review in five case studies, highlighting the key role these two methods play in advancing dynamic structural biology.
Curr Opin Struct Biol
· 2026 Jun · PMID 42275977
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Understanding how tumor cells navigate crowded spatial environments to drive progression and how to deter them is challenging. Intracellularly, dynamic protein ensembles link genotype to phenotype. Dysregulation of these...Understanding how tumor cells navigate crowded spatial environments to drive progression and how to deter them is challenging. Intracellularly, dynamic protein ensembles link genotype to phenotype. Dysregulation of these ensembles-driven by overexpression and mutational variants-alters the conformational landscapes, shifts cell states, and reshapes cell fate decisions. This diversity, spanning from the molecular level to the tumor microenvironment, triggers resistance mechanisms precipitating efforts to engineer effective combination strategies. Here, we underscore these transient cell states in migration and tissue adaptation, which depend on transcriptomic and signaling compatibility. Our spatial biology outlook envisions a map for designing drug combination strategies targeting both the primary tumor and disseminating cell states with host tissue commonalities, centering on bypass pathways to deter drug resistance and metastasis.
The molecular basis for specific pairing between transcriptional enhancers and promoters remains a highly active area of investigation, even over four decades after enhancers were first discovered. Numerous studies have...The molecular basis for specific pairing between transcriptional enhancers and promoters remains a highly active area of investigation, even over four decades after enhancers were first discovered. Numerous studies have explored general mechanisms such as those involving the cohesin/CTCF system and the core transcriptional machinery, including RNA polymerase 2, but these fail to account for the specificity of gene expression across cell types, maturation stages, and cell cycle intervals. Genetic loss of tissue-specific transcription factors provided early insights, but the findings were fraught with potentially confounding secondary effects. The advent of tools permitting rapid protein degradation has changed that. Here we discuss recent studies of illustrative mammalian transcription (co-) factors with architectural roles that contribute to enhancer-promoter communication, and contextualize their functions within generic chromatin-organizing principles.
Neurodegeneration has traditionally been largely attributed to protein aggregation, yet ribonucleic acid (RNA) has emerged as an active driver of pathology. Expanded repeat RNAs, misregulated RNA-binding proteins, and ab...Neurodegeneration has traditionally been largely attributed to protein aggregation, yet ribonucleic acid (RNA) has emerged as an active driver of pathology. Expanded repeat RNAs, misregulated RNA-binding proteins, and aberrant RNA-protein interactions can directly or indirectly trigger neuronal dysfunction, although the distinction between the two mechanisms might, in some cases, be loose. RNA modulates prion-like aggregation, scaffolds liquid-liquid phase separation, and either promotes or inhibits protein assembly, depending on RNA sequence and structure. The aim of this review is to discuss our current understanding of RNA's dual role-as a facilitator of aggregation or as a potential therapeutic target-revealing new mechanistic insights into diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and spinocerebellar ataxias. We highlight RNA metabolism as a central determinant of neuronal vulnerability.
Glycosylation can be critical for determining the structure and functions of proteins, but it is often neglected, leading to significant knowledge gaps in our understanding of biology. The inherent heterogeneity of glyca...Glycosylation can be critical for determining the structure and functions of proteins, but it is often neglected, leading to significant knowledge gaps in our understanding of biology. The inherent heterogeneity of glycans presents technical challenges to glycoprotein characterisation and impedes the representation of intact glycoprotein structures. Here we discuss how glycan heterogeneity constitutes a fundamental property of glycoproteins and acts as a remarkably powerful strategy for modulating biological function on the fly, complementing the rigidity of the genome. We present recent examples of how integrating mass spectrometry glycoproteomics and glycomics, structural biology, and molecular dynamics simulation data can bring glycans into a 3D structural framework, providing a unique perspective into the roles of glycosylation and potentially accelerating the design of glycoprotein biologics. Strengths and limitations of these approaches are also highlighted.
Sugar transporters are essential for the long-distance carbon allocation in plants and contribute to both biotic and abiotic stress responses yet the molecular mechanisms of substrate specificity and conformational cycli...Sugar transporters are essential for the long-distance carbon allocation in plants and contribute to both biotic and abiotic stress responses yet the molecular mechanisms of substrate specificity and conformational cycling are only beginning to emerge. Recent structural and biophysical studies of the Sugar Transport Protein, the Sucrose Carrier, and the Sugar Will Eventually be Exported Transporter families have revealed how both conserved and divergent features shape substrate recognition and conformational cycling. Despite these advances, complete transport cycles and native substrate-bound states remain unresolved for several transporters. Advancing our understanding will require integrating structural, dynamic, and functional insights to define the mechanisms that govern sugar transport which could enable future engineering of transport proteins relevant for plant resilience and productivity.
High-speed atomic force microscopy (HS-AFM) enables direct nanometer-resolution visualization of single molecules and molecular assemblies in real-time and under physiological conditions, providing unique insights into h...High-speed atomic force microscopy (HS-AFM) enables direct nanometer-resolution visualization of single molecules and molecular assemblies in real-time and under physiological conditions, providing unique insights into how membranes and membrane proteins move and interact within native lipid environments. Recent methodological advances and integration with complementary techniques have extended HS-AFM to increasingly complex, physiologically relevant systems, bridging gaps between high-resolution static structural methods and low-resolution functional dynamics. Here, we highlight how HS-AFM has changed our understanding of membrane organization, protein conformational dynamics, and lipid-protein coupling. By capturing transient events inaccessible to ensemble approaches, HS-AFM is transforming our ability to connect structural snapshots with functional behavior, advancing dynamic structural biology.
Cryo-electron tomography (cryo-ET) enables the analysis of biological samples in situ, revealing the complex interplay that shapes (sub)cellular architecture. Across the cryo-ET workflow, diverse forms of contextual info...Cryo-electron tomography (cryo-ET) enables the analysis of biological samples in situ, revealing the complex interplay that shapes (sub)cellular architecture. Across the cryo-ET workflow, diverse forms of contextual information-from experimental metadata to spatial organization and particle-specific annotations-can inform the experimental design, guide computational approaches, and deepen the interpretation of tomograms. In this review, we highlight recent advances in contextual analysis that complement and enhance commonly used cryo-ET workflows, while also expanding their scope. Examples range from sample preparation to data analysis aided by molecular dynamics simulations. Together, they illustrate how different notions of context enrich in situ investigations and allow cryo-ET to extend beyond high-resolution structure determination toward a more comprehensive understanding of cellular environments.
Cryogenic electron tomography (cryoET) offers unparalleled views into the molecular architecture of cells. As no stains or fixation are used, electrons scatter off the native atoms, and all molecules contribute to the fi...Cryogenic electron tomography (cryoET) offers unparalleled views into the molecular architecture of cells. As no stains or fixation are used, electrons scatter off the native atoms, and all molecules contribute to the final tomogram. As a result, it can be challenging to identify proteins of interest, especially inside a crowded cellular environment. Recent developments in molecular tags for cryoET provide several options for identifying proteins in reconstructed tomograms, but these are often not appropriate for finding an area of interest when collecting data. To increase the utility and throughput of cryoET, future approaches should combine correlative light and electron microscopy (CLEM) with tagging, so that a single modification can be used at small and large spatial scales. Automation of the detection of tags in tomograms and correlation between imaging modalities using machine learning methods will help increase the throughput of these methods, making them more suitable for rare events or structure determination by sub-tomogram averaging.