While protein characterisation is critical for continuing drug discovery and development, many techniques fall short of capturing the range of conformational dynamics that underpin the targeted protein activity. Hydrogen...While protein characterisation is critical for continuing drug discovery and development, many techniques fall short of capturing the range of conformational dynamics that underpin the targeted protein activity. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for investigation of protein structural dynamics, particularly for membrane proteins in different mimetic environments. This measurement of intrinsic protein behaviour is invaluable in outlining pathogenic protein dynamics, protein-ligand profiles, and druggable protein centres that are often inaccessible in other structural techniques. This minireview will cover how recent advances have been applied to HDX-MS of membrane proteins and peptides widening the use of HDX for drug discovery.
Curr Opin Struct Biol
· 2025 Aug · PMID 40472449
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As cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) continue to advance, the ability to visualize cellular and organismal structures with unprecedented clarity is redefining the landscape of stru...As cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) continue to advance, the ability to visualize cellular and organismal structures with unprecedented clarity is redefining the landscape of structural biology. Breakthroughs in imaging technology, sample preparation and image processing now enable the detailed elucidation of cellular architecture, macromolecular organization, and dynamic biological processes at sub-nanometer resolution. Recent methodological advances have propelled the field to new frontiers, facilitating the investigation of complex biological questions across scales-from macromolecular complexes to organism-wide structural insights. This review explores rapidly emerging trends, highlights key innovations that are pushing the boundaries of in situ structural biology, and addresses persistent challenges in expanding the applicability of cryo-EM and cryo-ET across diverse biological systems.
Detergent micelles are widely used as a membrane-mimetic system for membrane protein extraction, isolation and structural study. Many recently developed detergents feature multiple tail and head groups, with architecture...Detergent micelles are widely used as a membrane-mimetic system for membrane protein extraction, isolation and structural study. Many recently developed detergents feature multiple tail and head groups, with architectures that are symmetric (i.e. the same alkyl chain) and non-hybrid (single head group type). Further diversification has involved incorporating structural differences in the tail groups (unsymmetric), head groups (hybrid), or both head and tail groups (unsymmetric hybrid). In this mini-review, we introduce these novel detergents, focusing on the relationships between their structural features, physical properties and performance in membrane protein applications. The detergent design strategy utilizing unsymmetric/hybrid structures expands the detergent repertoire and the detergent structure-property-efficacy relationships presented offer valuable design guidelines, collectively advancing membrane protein research.
Reconstitution of membrane proteins in nanodiscs has proven to be a highly effective approach to study membrane protein structures in a lipid bilayer, resulting in many recent single particle cryo-EM structures. While mo...Reconstitution of membrane proteins in nanodiscs has proven to be a highly effective approach to study membrane protein structures in a lipid bilayer, resulting in many recent single particle cryo-EM structures. While most of these studies employed membrane scaffold protein (MSP) nanodiscs, additional types of nanodiscs were developed based on MSPs and provide alternative approaches. Nanodiscs have been particularly effective in solving structures of different protein conformations and of bound lipids, demonstrating key roles of specific lipids in structural integrity and protein function. At the same time, discrepancies of lipid behavior in nanodiscs compared to native membranes and liposomes necessitate careful scrutiny of reconstitution parameters and further evaluation. This brief review covers an overview of types of nanodiscs currently in use for cryo-EM structural studies, their advantages and limitations, as well as examples of the dramatically increased understanding they can reveal.
Trebesch N, Hasdemir HS, Chen T
… +2 more, Wen PC, Tajkhorshid E
Curr Opin Struct Biol
· 2025 Aug · PMID 40424866
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Membranes are fundamental components of cells that are involved in a wide variety of cellular functions. They are inherently complex, being composed of hugely diverse collections of lipids and proteins, and their various...Membranes are fundamental components of cells that are involved in a wide variety of cellular functions. They are inherently complex, being composed of hugely diverse collections of lipids and proteins, and their various functions arise directly from the intricate interplay between their components. To investigate the interactions between these components in detail, molecular dynamics (MD) simulations have proven to be an invaluable tool. In this mini-review, we highlight several recent studies that illustrate the current state of the art in using MD to study membrane systems. In particular, we first examine how MD is being used to characterize membrane binding of peripheral membrane proteins, we next describe how interactions between lipids and integral membrane proteins are being probed with MD, and we conclude by discussing new tools that have recently been developed to address the significant challenge of building simulateable models of large-scale membranes with complex curvature.
Although membrane proteins constitute a significant portion of the genomes of all species and represent well-validated targets for numerous therapeutic interventions, high-resolution structural knowledge of this class of...Although membrane proteins constitute a significant portion of the genomes of all species and represent well-validated targets for numerous therapeutic interventions, high-resolution structural knowledge of this class of proteins still falls behind that of their soluble counterparts. Despite serious technological developments in the methods presently available for structural characterizations, as well as decades spent on such investigations, membrane proteins remain notoriously difficult to study. This is particularly true for environments which mimic native membranes well enough to maintain their proper functional states. This mini review covers the most recent advances in the structural and dynamic characterization of membrane proteins through the utilization of solution nuclear magnetic resonance methods applied to lipid nanodiscs.
In recent years, the application of natural language models to protein amino acid sequences, referred to as protein language models (PLMs), has demonstrated a significant potential for uncovering hidden patterns related...In recent years, the application of natural language models to protein amino acid sequences, referred to as protein language models (PLMs), has demonstrated a significant potential for uncovering hidden patterns related to protein structure, function, and stability. The critical functions of proteins in biological processes often arise through interactions with small molecules; central examples are enzymes, receptors, and transporters. Understanding these interactions is particularly important for drug design, for bioengineering, and for understanding cellular metabolism. In this review, we present state-of-the-art PLMs and explore how they can be integrated with small molecule information to predict protein-small molecule interactions. We present several such prediction tasks and discuss current limitations and potential areas for improvement.
Curr Opin Struct Biol
· 2025 Aug · PMID 40381356
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Cryo-electron tomography (cryo-ET) is an advanced and rapidly evolving imaging technique that enables three-dimensional visualization of biological structures in their native state. Although cryo-ET has historically face...Cryo-electron tomography (cryo-ET) is an advanced and rapidly evolving imaging technique that enables three-dimensional visualization of biological structures in their native state. Although cryo-ET has historically faced significant challenges, including limited applications, tedious data acquisition, labor-intensive image processing, and lower resolution when compared with single particle cryo-electron microscopy (cryo-EM), recent breakthroughs in hardware and software development have significantly improved the entire cryo-ET workflow to enable higher throughput and resolution. These advances have accelerated discoveries in structural and cellular biology, particularly in microbiology, where cryo-ET has unveiled unprecedented insights into the inner life of microbes. This review presents pivotal advances propelling high-throughput cryo-ET and the visualization of microbial architecture. As innovations in imaging technologies, workflow automation, and computational methods continue progressing rapidly, cryo-ET is expected to be increasingly utilized across various fields of life sciences, shaping the future of biological research and biomedical discoveries.
The biology of gene regulation in eukaryotic genomes is a mature field. The biochemical principles of factor binding to DNA are well-known from in vitro studies, as are the structural interactions in which specific domai...The biology of gene regulation in eukaryotic genomes is a mature field. The biochemical principles of factor binding to DNA are well-known from in vitro studies, as are the structural interactions in which specific domains of these proteins interface across a short stretch of DNA to confer sequence-specific recognition. Whereas the basic principles of binding and dissociation defined in vitro apply in vivo, the living nucleus is a dynamic compartment crowded with molecules, including motors that drive chromatin movements critical for the regulation of gene expression. Understanding these dynamics in vivo has spurred the development of cutting-edge technologies to observe factor-DNA interactions. The biological significance of chromatin dynamics is now revealed by a wide variety of high-resolution chromatin profiling methods.
Cryo-electron tomography is the best-suited imaging technique for visual proteomics. Recent advances have increased the number, quality, and resolution of tomograms. However, object detection is the bottleneck task of th...Cryo-electron tomography is the best-suited imaging technique for visual proteomics. Recent advances have increased the number, quality, and resolution of tomograms. However, object detection is the bottleneck task of the analysis workflow because, so far, only a few molecules can be detected by computer methods for pattern recognition. This article introduces the major challenges in detecting molecular complexes for cryo-electron tomography. This paper also identifies the limitations of the current methods. Finally, it describes the approaches proposed to overcome these limitations.
Reconstitution and direct extraction of membrane proteins using saposins is an emerging technique for solubilizing and stabilizing membrane proteins. The Salipro technology offers several advantages over traditional dete...Reconstitution and direct extraction of membrane proteins using saposins is an emerging technique for solubilizing and stabilizing membrane proteins. The Salipro technology offers several advantages over traditional detergent solubilization, including a more native lipid environment, increased protein stability, and maintenance of functionality. This review covers recent studies that have used Salipros to characterize membrane proteins, as well as advances in direct extraction methods that have enabled the structural and functional characterization of a variety of targets.
Liu ZH, Tsanai M, Zhang O
… +2 more, Head-Gordon T, Forman-Kay JD
Curr Opin Struct Biol
· 2025 Aug · PMID 40349675
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Intrinsically disordered proteins and regions are increasingly appreciated for their abundance in the proteome and the many functional roles they play in the cell. In this short review, we describe a variety of approache...Intrinsically disordered proteins and regions are increasingly appreciated for their abundance in the proteome and the many functional roles they play in the cell. In this short review, we describe a variety of approaches used to obtain biological insight from the structural ensembles of disordered proteins, regions, and complexes and the integrative biology challenges that arise from combining diverse experiments and computational models. Importantly, we highlight findings regarding structural and dynamic characterization of disordered regions involved in binding and phase separation, as well as drug targeting of disordered regions, using a broad framework of integrative modeling approaches.
Curr Opin Struct Biol
· 2025 Jun · PMID 40349511
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Chromatin, the complex of DNA and proteins that organises genetic material in eukaryotic cells, has been a focal point of biological research for over a century. Its structure determines critical functions such as gene r...Chromatin, the complex of DNA and proteins that organises genetic material in eukaryotic cells, has been a focal point of biological research for over a century. Its structure determines critical functions such as gene regulation, DNA replication and chromosome segregation. Early models of chromatin were limited by technological constraints, but advancements in imaging, particularly X-ray and electron microscopy (EM), gradually unveiled its hierarchical organisation. The recent emergence of cryo-electron tomography (cryo-ET) coupled with cryo-focused ion beam (cryo-FIB) milling has revolutionised our understanding of chromatin organisation by providing native, three-dimensional (3D) views of various macromolecules and architectures of chromatin at unprecedented resolution. This review traces the historical progression of chromatin structural studies, from early EM and fluorescence microscopy to the transformative insights offered by cryo-ET, culminating in a synthesis of current knowledge and future directions.
Curr Opin Struct Biol
· 2025 Jun · PMID 40344741
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Understanding genome organization at the kilobase to megabase scale is critical, as it encompasses genes and regulatory elements. Improvements in the resolution of experimental techniques have revealed novel structural m...Understanding genome organization at the kilobase to megabase scale is critical, as it encompasses genes and regulatory elements. Improvements in the resolution of experimental techniques have revealed novel structural motifs at this scale, including micro-compartments, nucleosome clutches, microdomains, and packing domains. Here we review recent progress on developing theories to explain these observations. Key advances include elucidating the role of nucleosome positioning and epigenetic modifications, the role and mechanisms of compartmentalization in local structure, and the interplay between loop extrusion and phase separation. This work has revealed probable mechanisms by which the observed structures emerge, but it remains unclear how these factors act together in the cell. To this end, recent studies have used chromatin conformation capture data in concert with diverse genomics datasets to create native-like models of chromatin at nucleosome resolution and below. While several roadblocks remain, this strategy promises to decode how molecular forces sum to shape chromatin structure and ultimately regulate transcription.
Eukaryotic cell divisions pass on genetic and epigenetic information from parental to daughter cells through replication of the chromatin, which needs to be reestablished following DNA replication, as its building block,...Eukaryotic cell divisions pass on genetic and epigenetic information from parental to daughter cells through replication of the chromatin, which needs to be reestablished following DNA replication, as its building block, the nucleosome, is disrupted by the passage of the DNA replication fork. This replication-coupled (RC) nucleosome assembly process takes place in distinct pathways depending on whether newly synthesized or parental histones are used. This review highlights recent progress in structural and biochemical studies of RC nucleosome assembly, focusing on the roles of histone chaperones in both de novo assembly of nucleosomes from newly synthesized histones and the recycling of parental histones. We also discuss the interactions between histone chaperones and replisome components that govern the coupling of nucleosome assembly to chromatin replication. Finally, we offer our perspective on future efforts in advancing this important research direction.
Overduin M, Bhat R, Dieudonné T
… +2 more, Zhang P, Kervin TA
Curr Opin Struct Biol
· 2025 Jun · PMID 40339327
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Each cell possesses a genetic and a proteolipid code that together convey molecular information in a perpetual cycle. One element of this cycle is the recognition of lipids that work together to specify subcellular locat...Each cell possesses a genetic and a proteolipid code that together convey molecular information in a perpetual cycle. One element of this cycle is the recognition of lipids that work together to specify subcellular locations for biochemical activity. These "lipidons" are now being resolved in protein structures from eukaryotic plasma membranes, endosomes, mitochondria, prokaryotes, and viruses with technologies like in situ cryo-electron imaging and membrane-active polymers. This adds to an expanding catalogue of codified protein-lipid interactions that are recontextualizing cell biology and drug discovery.
The NOD-like receptor (NLR) family comprises inflammasome sensors that are critical intracellular pattern recognition receptors of the innate immune system. The NLR family members NLRP3 and NLRP1 can be activated by a wi...The NOD-like receptor (NLR) family comprises inflammasome sensors that are critical intracellular pattern recognition receptors of the innate immune system. The NLR family members NLRP3 and NLRP1 can be activated by a wide range of pathogenic, chemical, self-derived and stress-related stimuli. In recent years, remarkable progress in functional and structural studies of these two receptors have shed light on their complicated and entirely different activation and regulation mechanisms. This review focuses on recent structural studies of NLRP3 and NLRP1, emphasizing the regulatory steps mediated by various activation and inhibitory factors.
AAA+ proteins are a large family of ATPases involved in a myriad of cellular activities. Recent advances in AAA+ proteins, especially cryoEM structures of these proteins in complex with their substrates, have provided ke...AAA+ proteins are a large family of ATPases involved in a myriad of cellular activities. Recent advances in AAA+ proteins, especially cryoEM structures of these proteins in complex with their substrates, have provided key insights into how they function. Here we review recent progress in structural studies and mechanistic understanding of AAA+ proteins involved in DNA processing, including gene transcription, DNA replication, repair/recombination and transposition. Using a few selected examples, we show how AAA+ proteins act on both DNA and protein peptides, which are often enclosed in the pores of AAA+ hexamers. We propose that using AAA+ proteins to translocate a peptide to partially unfold a substrate is an effective strategy in disassembling an assembled complex. Further, several studies show that although they often act as asymmetric hexamers in their active form, AAA+ proteins adopt a range of oligomers for their functions.
Molecular glues are small drug-like molecules that induce de novo protein-protein interactions or facilitate pre-existing weak interactions between proteins. In the context of a ubiquitin ligase, such binding events freq...Molecular glues are small drug-like molecules that induce de novo protein-protein interactions or facilitate pre-existing weak interactions between proteins. In the context of a ubiquitin ligase, such binding events frequently result in ubiquitination by proximity. Rational development of these transformative modalities, however, remains a major challenge. Here we review recent insights into molecular glues and the emerging design principles. Protein surfaces can similarly be complemented by mutations or compounds inducing binding and a resulting gain of functionality. When the interaction surface between two proteins is relatively small, or when the affinity between the proteins is otherwise weak, proportionally more binding energy will have to be provided by the compound to glue the proteins together. We suggest a simple thermodynamic model to rationalize molecular glue action facilitated by compounds and mutations.
Curr Opin Struct Biol
· 2025 Jun · PMID 40334519
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Autonomous non-long-terminal repeat (non-LTR) retrotransposons, including long interspersed elements (LINEs), are mobile genetic elements abundant in eukaryotic species that shape the genomic landscape and host physiolog...Autonomous non-long-terminal repeat (non-LTR) retrotransposons, including long interspersed elements (LINEs), are mobile genetic elements abundant in eukaryotic species that shape the genomic landscape and host physiology in both health and disease. Non-LTR retrotransposons create new genomic copies through a mechanism termed target-primed reverse transcription, where the retrotransposon-encoded protein nicks target DNA to prime reverse transcription templated by bound RNA, typically its own encoding mRNA. Until recently, structural information on non-LTR retrotransposons was lacking due to challenges in purification and reconstitution of active complexes. Recent biochemical studies and cryo-electron microscopy structures of complexes from insect, bird, and turtle site-specific R2 retrotransposons and the human LINE-1 retrotransposon have provided important insights. Here we discuss these studies and their implications for retrotransposon evolution and eukaryotic genome biology.