This article was generated entirely by AI without human authorship. Please read with discretion, or have an AI verify its accuracy.
AI TranslationSimplified ChineseEnglish
Key Insights
This issue brings together 54 papers covering GPCR signaling regulation, molecular labeling and spatial omics, cryo-EM methods, ion channels and neural activity, protein design, chloroplast protein import, CTP synthesis, targeted protein degradation, and more.
GPCR Recognition, Dynamics, and Signaling Regulation (17 papers)
01 · An opioid efficacy switch for reversible optical control of analgesia
Cell · Publication/issue date: 2026-10-15
Authors: Luca Posa, Giovanna R. Romano, Xiang Ji, Saif Khan, Antonina L. Nazarova, Sheida Sharghi Moshtaghin, Bruno Paz, Gye Won Han, Anisul Arefin, Alberto J. Gonzalez-Hernandez, Ipsit Srivastava, Saheem A. Zaidi, Kenny Calderon-Godinez, Zixin Zhang, Aaliyah S. Tyson, Mohsen Ranjbar, Kristen E. Pleil, Kanupriya Pande, Vsevolod Katritch, Cornelius Gati, Dirk Trauner, Joshua Levitz
The μ-opioid receptor is a major target for pain relief, but systemic dosing makes it difficult to restrict where a drug acts or separate analgesia from adverse effects. This study developed AM-3, a reversibly photoswitchable agonist that controls receptor signaling by switching between low- and high-efficacy states under illumination. Structures of receptor complexes in the two states reveal distinct binding modes, accompanied by differences in ligand, receptor, and G protein dynamics. In mouse pain models, AM-3 produced reversible, repeatable, light-controlled antinociception. The authors link its reduced adverse effects to limited brain accumulation and the ability to activate it locally with light. The work offers a new tool for studying receptor efficacy and localized analgesia, but the evidence so far comes from animal experiments and does not establish efficacy or safety in humans.
Highlight · Reversible photoswitching changes AM-3’s efficacy at the receptor, enabling localized, repeatable control of analgesia in mice.
Authors: Katarina Nemec, Bálint Mészáros, Georgios Skiniotis, Scott C. Blanchard, M. Madan Babu
Ligand binding and signaling by GPCRs are not a simple on–off switch, but a multilayered process spanning atomic contacts, receptor conformations, and cellular pathways. Moving through increasing levels of complexity, this review brings together recent research on how receptors recognize extracellular signals, undergo ligand-induced conformational changes, and interact with different signaling adaptors and intracellular pathways. It places particular emphasis on context: the behavior of a given receptor depends on its molecular partners, regulatory state, and disease environment, so static structures explain only part of the picture. The authors also discuss how these insights could guide more selective interventions. The review’s value lies in its multiscale framework linking structure to function, helping readers understand why findings at different experimental levels are connected but not directly interchangeable.
Highlight · Understanding GPCR pharmacology requires a unified view of ligand contacts, conformational changes, and cellular signaling.
03 · The Role of Intrinsically Disordered Domains in Regulating G Protein-Coupled Receptor Signaling
Journal of the American Chemical Society · Publication/issue date: 2026-01-14
Authors: Jun Xu, Ruoyi Qiu, Alexander M. Garces, Harald Hübner, Xinyu Xu, Chengyu Hao, Dorothee Weikert, Peter Gmeiner, Michael T. Lerch, Brian K. Kobilka
Signaling by the α2A-adrenergic receptor is shaped not only by its transmembrane core but potentially also by its flexible, disordered regions. Using multiple single-molecule FRET labeling configurations to track conformational changes in real time, the researchers found a high energy barrier to the outward movement of TM6 required for activation, while the partially disordered extracellular loop ECL2 showed ligand-specific dynamics. The long third intracellular loop, ICL3, adopted a relatively compact conformation close to the membrane in the basal state and negatively modulated coupling to signaling transducers through an allosteric mechanism. Its multiple functional substates were also influenced by kinase phosphorylation and drug efficacy. These findings recast disordered loops as dynamic regulatory modules rather than passive linkers, helping explain how the same receptor can produce distinct signaling profiles in response to different ligands.
Highlight · Dynamic conformations of disordered GPCR loops can regulate ligand efficacy and coupling to signaling proteins.
04 · Single-molecule analysis of ligand efficacy in β2AR–G-protein activation
Nature · Publication/issue date: 2017-07
Authors: G. Glenn Gregorio, Matthieu Masureel, Daniel Hilger, Daniel S. Terry, Manuel Juette, Hong Zhao, Zhou Zhou, Jose Manuel Perez-Aguilar, Maria Hauge, Signe Mathiasen, Jonathan A. Javitch, Harel Weinstein, Brian K. Kobilka, Scott C. Blanchard
Differences in GPCR ligand efficacy may arise from distinct dynamics during receptor and G protein activation. This study used single-molecule FRET to monitor TM6 movement in the β2-adrenergic receptor, comparing ligands of different efficacies in the presence and absence of Gs. Partial and full agonists modulated TM6 in different ways, affecting both the rate of GDP-bound receptor–Gs complex formation and the efficiency of subsequent nucleotide exchange. The experiments also captured transient nucleotide-bound states not adequately represented by existing static structures. By tracing the allosteric connection between the ligand-binding pocket and the G protein’s nucleotide-binding pocket through these dynamic processes, the work suggests that drug efficacy depends not just on an end-state conformation, but also on the rates and pathways of transitions between states.
Highlight · Ligands produce different levels of Gs activation by altering receptor motions and nucleotide exchange kinetics.
05 · Ligand efficacy modulates conformational dynamics of the µ-opioid receptor
Nature · Publication/issue date: 2024-05
Authors: Jiawei Zhao, Matthias Elgeti, Evan S. O’Brien, Cecília P. Sár, Amal EI Daibani, Jie Heng, Xiaoou Sun, Elizabeth White, Tao Che, Wayne L. Hubbell, Brian K. Kobilka, Chunlai Chen
The μ-opioid receptor is a major target for analgesic drugs, and understanding how ligands produce different intrinsic efficacies requires a view of conformational dynamics rather than a single structure. Combining double electron–electron resonance with single-molecule FRET, the researchers identified multiple states of the receptor’s cytoplasmic side that interconvert on different timescales. A preactivated conformation could bind G proteins, whereas a fully activated conformation substantially reduced GDP affinity in the ternary complex, showing that binding and productive activation are distinct steps. The authors also compared how β-arrestin-1 and Gi interact with the receptor core, finding lower affinity and weaker specificity for β-arrestin-1. These results link ligand-specific conformational distributions to downstream efficacy, providing a dynamic basis for analyzing opioid receptor signaling selectivity.
Highlight · Preactivated and fully activated states of the μ-opioid receptor play distinct roles in G protein binding and GDP release.
Authors: Jonathan C. Deutsch, Daniel Hilger, John Janetzko, Charles M. Schroeder, Steven Chu, Brian K. Kobilka, Rabindra V. Shivnaraine
Following receptor activation, the G protein’s α-helical domain must open to expose the nucleotide-binding site and allow nucleotide exchange. This study used single-molecule FRET to directly observe Gi coupled to the μ-opioid receptor, comparing domain motions across different ligands and nucleotide states. The results show that ligands bound at a distant site on the receptor can influence G protein dynamics, with higher-efficacy agonists more readily driving the protein into open states with low nucleotide affinity. The experiments also captured transient intermediates during nucleotide exchange. By extending the study of drug action beyond the receptor to its coupling partner, the work shows that allosteric modulation of the G protein’s conformational ensemble is another mechanism that encodes opioid ligand efficacy, rather than a wholly passive consequence of receptor changes.
Highlight · Differences in opioid ligand efficacy can propagate to Gi domain motions and nucleotide exchange.
Authors: Marina Casiraghi, Haoqing Wang, Patrick C. Brennan, Chris Habrian, Harald Hübner, Maximilian F. Schmidt, Luis Maul, Biswaranjan Pani, Sherif M. F. M. Bahriz, Bing Xu, Nico Staffen, Tufa E. Assafa, Bohan Chen, Elizabeth White, Roger K. Sunahara, Asuka Inoue, Yang K. Xiang, Robert J. Lefkowitz, Ehud Y. Isacoff, Nathaniel Nucci, Peter Gmeiner, Michael T. Lerch, Brian K. Kobilka
The β2-adrenergic receptor generally favors Gs and couples more weakly to Gi, but the mere presence or absence of a complex cannot explain this selectivity. After identifying the Gi-biased agonist LM189, the researchers combined structural and biophysical evidence to compare the receptor states required for coupling to different G proteins. Their results indicate that specific conformations of intracellular loop ICL2 and transmembrane helix TM6 work together to distinguish Gs from Gi, with receptor dynamics also contributing to this preference. The work thus links G protein selectivity to variable conformational features within the receptor, rather than treating it as a fixed property of its sequence. This provides a mechanistic basis for understanding biased agonists and highlights the need to consider both structural states and functional measurements when assessing signaling selectivity.
Highlight · Conformational differences in ICL2 and TM6 jointly influence the β2 receptor’s preference for coupling to different G proteins.
Authors: Annika Ullrich, Johannes Schneider, João M. Braz, Eduard Neu, Nico Staffen, Markus Stanek, Jana Bláhová, Tamsanqa Hove, Tamara Albert, Anni Allikalt, Stefan Löber, Karnika Bhardwaj, Sian Rodriguez-Rosado, Elissa Fink, Tim Rasmussen, Harald Hübner, Asuka Inoue, Brian K. Shoichet, Allan I. Basbaum, Bettina Böttcher, Dorothee Weikert, Peter Gmeiner
The 5-HT1A receptor participates in pain regulation, but how to harness signaling bias to achieve a desirable pharmacological profile remains an important research question. Starting from a compound library, the authors identified the bitopic agonist ST171, which primarily activated Gi/o, showed no Gs activation and weak β-arrestin recruitment, and had effects in acute and chronic pain models. Cryo-EM comparisons of the ST171 complex with Gi and Gs complexes of the conventional agonist befiradol showed that the ligands occupy different peripheral sites and are associated with distinct receptor conformations. Molecular dynamics simulations helped explain these differences. The study connects binding modes, signaling selectivity, and analgesic effects in experimental models, but these remain mechanistic and preclinical findings; they do not directly establish efficacy or a safety advantage in humans.
Highlight · Distinct peripheral binding modes and receptor conformations provide a structural explanation for signaling bias among 5-HT1A agonists.
Authors: Geng Chen, Jana Bláhová, Nico Staffen, Harald Hübner, Nadja Nunhöfer, Chen Qiu, Peter Gmeiner, Dorothee Weikert, Yang Du, Jun Xu
GPCR dimerization can alter how drugs act, and GPR3 offers an example of dimer-specific modulation. The study found that GPR3 forms constitutive homodimers in living cells, with the inhibitor AF64394 acting on this state as a negative allosteric modulator. Cryo-EM structures of the inhibitor-bound dimer and its Gs complex place the ligand at the transmembrane dimer interface. The authors propose that the ligand prevents the receptors from dissociating upon engagement with Gs and constrains the relevant transmembrane regions in an inactive-like intermediate conformation, thereby reducing Gs coupling. Together, the structural and functional findings show that receptor oligomerization can confer pharmacological selectivity and underscore the need to consider receptor–receptor interactions when studying allosteric inhibition.
Highlight · The GPR3 dimer interface offers a route to negative allosteric modulation distinct from conventional ligand-binding pockets.
10 · A TM7 conformational switch governs GPCR transducer selectivity
Preprint · Publication/issue date: 2026/04/21
Authors: Guodong He, Maik Pankonin, Guojing Yu, Haiyan Xu, Huan Li, Maximilian Salomon, Hossein Batebi, Xue Guo, Shuhao Zhang, Fang Kong, Li Zhan, Xiaoou Sun, Qingqing Ma, Lan Hua, Sheng Ding, Chuangye Yan, Xin Chen, Zhaobing Gao, Peter W. Hildebrand, Xiangyu Liu
This preprint examines how the μ-opioid receptor selects among different signaling partners. The authors identified an agonist with pronounced G protein bias and determined cryo-EM structures of its receptor–Gi and receptor–GRK2 complexes. Combining these structures with functional and dynamics data, they propose a stepwise gating model. In this model, conformational changes in TM7 act as a final checkpoint that can influence subsequent β-arrestin recruitment: even after completing some activation steps, the receptor need not proceed along every signaling pathway. The work frames biased pharmacology as selection within a multistep process, rather than simply a difference between active conformations. The evidence remains limited to the structural and experimental systems reported in this preprint and does not establish that the agonist offers a clinical safety advantage.
Highlight · TM7 dynamics may act as a final checkpoint in determining which signaling partners a GPCR engages.
Receptors in the neuropeptide Y family play distinct roles in appetite and mood regulation, and even small changes in a ligand can alter receptor selectivity. The study determined cryo-EM structures of the Y2 receptor–Gi complex bound to either PYY(3–36) or NPY. Together with cell-based experiments and simulations, these structures explain why PYY lacking its first two amino acids can still act effectively on Y2 but cannot similarly activate Y1. Comparing downstream pathways, the authors further found that Y2 favors G protein signaling upon activation, whereas Y1 did not show the same preference between G protein and β-arrestin signaling. The work links differences in ligand recognition to signaling bias, suggesting that even receptors within the same family cannot simply be assumed to share the same models of ligand binding and function.
Highlight · Both its ligand recognition features and its preference for G protein signaling distinguish Y2 from the related Y1 receptor.
Authors: Chaehee Park, Jinuk Kim, Seung-Bum Ko, Yeol Kyo Choi, Hyeongseop Jeong, Hyeonuk Woo, Hyunook Kang, Injin Bang, Sang Ah Kim, Tae-Young Yoon, Chaok Seok, Wonpil Im, Hee-Jung Choi
NPY participates in many physiological processes, including feeding and anxiety, but a complete structural account of its interactions with the Y1 receptor had been lacking. The study resolved the NPY–Y1–Gi1 complex, showing that the peptide’s extended C terminus enters the receptor’s transmembrane core, with its amidated terminal residue sitting at the bottom of the binding pocket. Meanwhile, the helical and N-terminal regions of NPY contact the receptor’s extracellular loops, further contributing to high-affinity binding. Structural analysis and mutagenesis together support this coordinated, multiregion recognition model. The findings help explain why different parts of the intact peptide can influence activity and provide a basis for comparing selectivity across Y1 and other NPY receptors, rather than attributing all pharmacological properties to a single local contact.
Highlight · NPY binding within the transmembrane pocket and its contacts with extracellular loops jointly support Y1 receptor recognition and activation.
13 · Structure-based discovery of nonopioid analgesics acting through the α2A-adrenergic receptor
Science · Publication/issue date: 2022-09-30
Authors: Elissa A. Fink, Jun Xu, Harald Hübner, Joao M. Braz, Philipp Seemann, Charlotte Avet, Veronica Craik, Dorothee Weikert, Maximilian F. Schmidt, Chase M. Webb, Nataliya A. Tolmachova, Yurii S. Moroz, Xi-Ping Huang, Chakrapani Kalyanaraman, Stefan Gahbauer, Geng Chen, Zheng Liu, Matthew P. Jacobson, John J. Irwin, Michel Bouvier, Yang Du, Brian K. Shoichet, Allan I. Basbaum, Peter Gmeiner
The α2A-adrenergic receptor is a potential target for nonopioid pain relief, but existing drugs often cause sedation. The researchers screened more than 300 million virtual molecules by docking to find new scaffolds distinct from those of commonly used drugs. They identified a set of active ligands, many of which showed partial agonism and a preference for Gi and Go signaling. Experimentally determined complex structures for two agonists supported the docking predictions and provided a basis for further optimization. Several initial hits and analogues produced target-dependent analgesia in multiple in vivo pain models without corresponding signs of sedation. The work demonstrates how large-scale structure-based screening can be combined with experimental validation and shows that ligands acting at the same receptor can differ in their pharmacological effects. These findings remain preclinical, however, and do not directly establish a therapeutic advantage in humans.
Highlight · New α2A agonists identified through structure-based screening suggest that analgesia may be separable from sedation in animal models.
14 · G protein-coupled receptors in neurodegenerative diseases and psychiatric disorders
Signal Transduction and Targeted Therapy · Publication/issue date: 2023-05-03
Authors: Thian-Sze Wong, Guangzhi Li, Shiliang Li, Wei Gao, Geng Chen, Shiyi Gan, Manzhan Zhang, Honglin Li, Song Wu, Yang Du
Neurodegenerative diseases and psychiatric disorders have complex, heterogeneous causes, and a single molecular change rarely explains the full range of symptoms. This review takes a GPCR-centered approach, summarizing the roles of different receptors in these conditions and how studies of receptor mechanisms and structures can create new opportunities for drug development. Focusing on emerging targets and recent advances in drug research, the authors place receptor-level insights in the broader context of multifactorial disease. The article offers an overview of targets and research directions; it does not imply that every receptor discussed has been validated to the same extent as a therapeutic target. Its value lies in connecting biological questions in neuropsychiatric disease with receptor systems that can be modulated, while acknowledging the evidence still needed to translate basic mechanisms into effective interventions.
Highlight · GPCRs offer multiple avenues for intervention in neuropsychiatric disease, but their value as targets must be assessed in light of disease heterogeneity.
15 · Cryo-EM structure of monomeric CXCL12-bound CXCR4 in the active state
Cell Reports · Publication/issue date: 2024-08-27
Authors: Yezhou Liu, Aijun Liu, Xinyu Li, Qiwen Liao, Weijia Zhang, Lizhe Zhu, Richard D. Ye
CXCL12–CXCR4 signaling contributes to hematopoietic cell localization and tumor-related processes, but the agonist-bound active structure had not been fully characterized. The study obtained a high-resolution cryo-EM structure of the CXCL12–CXCR4–Gi complex, showing one-to-one ligand–receptor binding consistent with the two-site model of chemokine recognition. The ligand’s N terminus enters the transmembrane pocket and makes contacts within the receptor that help drive activation. Structural, functional, and computational analyses together explain how these interactions connect to receptor conformational changes and G protein activation. By adding an active-state view to existing antagonist-bound structures, the work provides a foundation for understanding the receptor’s different pharmacological states, but does not directly establish a new clinical intervention.
Highlight · The CXCL12-bound active-state structure fills in the mechanistic picture of CXCR4 signaling, from ligand recognition to G protein activation.
16 · Photo-cross-linking-assisted deorphanization deciphers GPR50–L-LEN pairing in metabolism
Nature Chemical Biology · Publication/issue date: 2026-07
Authors: Rui Wu, Na Li, Zhihui Wen, Ya Wang, Shitian Li, Hao Zhou, Yuehong Huang, Xiao Xie, Xiaoyun Zhao, Xiaohui Wang, Shaoxin Tao, Guoliang Chai, Tongfei A. Wang, Zhen Li, Peng R. Chen, Miao Jing
Many GPCRs still lack known endogenous ligands, limiting studies of their physiological functions and pharmacology. The authors developed a photo-cross-linking-assisted receptor deorphanization platform that captures ligands at the recognition interface to identify them in native biological samples. They tested its performance on several known receptor–ligand pairs. The study further identifies the neuropeptide L-LEN as a proposed endogenous ligand for GPR50, with evidence that it regulates cellular activity through this receptor and downstream Gi signaling. In mice, this pairing is linked to brain–adipose communication, energy expenditure, and thermogenesis; loss of relevant components increases the likelihood of entering a torpor-like state under stress. The work provides both a tool for ligand discovery and clues to metabolic regulation, although its applicability across biological settings requires further investigation.
Highlight · Photo-cross-linking-assisted capture identifies a candidate endogenous ligand for GPR50 and links the receptor to body temperature and energy regulation.
17 · Synthetic GPCRs for programmable sensing and control of cell behaviour
Nature · Publication/issue date: 2025
Authors: Nicholas A Kalogriopoulos, Reika Tei, Yuqi Yan, Peter M Klein, Matthew Ravalin, Bo Cai, Ivan Soltesz, Yulong Li, Alice Y Ting
Synthetic receptors can translate specific antigens into cellular responses, but existing systems face limitations in the forms of antigen they recognize, the outputs they produce, and their amenability to drug control. The study introduces PAGER synthetic GPCRs, in which antigen binding relieves receptor inhibition, enabling a subsequent response to a drug and thus linking recognition to a controllable signaling output. The authors demonstrate responses to more than a dozen soluble and cell-surface antigens, with different receptor scaffolds driving gene expression, fluorescent readouts, or endogenous G protein activity. Applications include cell migration, differentiation, antibody secretion, and modulation of neural activity in brain slices. The work illustrates the versatility of modular receptor engineering, but validation across these settings does not mean that all corresponding therapeutic applications are ready for use; each still requires evaluation in its specific cellular context.
Highlight · PAGER combines antigen recognition, drug control, and multiple GPCR outputs in a programmable platform for cellular sensing.
Authors: Anneliese M. M. Gest, Sohum Mehta, Jin Zhang
Advances in fluorescent proteins and biosensors increasingly depend on the interplay between chemistry, physics, protein engineering, and screening technologies. This review surveys recent progress in fluorescent protein engineering, focusing on design goals such as improving photostability and shifting emission to longer wavelengths. Using mScarlet as a case study, it illustrates how insights from different fields can translate into better performance. The article also summarizes developments in fluorescent sensors for screening, quantitative imaging, spatial resolution, and multiplexed detection, considering how probes and measurement methods together expand what can be observed. Finally, the authors discuss directions for next-generation fluorescent tools. The article is a broad review of existing technologies and design approaches, rather than a performance report on a single new probe.
Highlight · Progress in fluorescent tools comes from coordinated advances in molecular design, screening, and imaging methods.
19 · Linking chromatin acylation mark-defined proteome and genome in living cells
Cell · Publication/issue date: 2023-03-02
Authors: Fangfei Qin, Boyuan Li, Hui Wang, Sihui Ma, Jiaofeng Li, Shanglin Liu, Linghao Kong, Huangtao Zheng, Rongfeng Zhu, Yu Han, Mingdong Yang, Kai Li, Xiong Ji, Peng R. Chen
Histone modifications are linked to metabolic state and can alter chromatin interactions and gene regulation, but directly connecting a modification at a specific site to its local environment is challenging. This study introduces SiTomics, a multiomics strategy with single-site resolution that profiles the proteins and genomic regions associated with specific chromatin acylation marks in living cells. Comparing crotonylation and β-hydroxybutyrylation following short-chain fatty acid stimulation, the authors found distinct association profiles and identified GLYR1 as an interacting protein that helps regulate H3K56cr localization within gene bodies. The results also linked some effects of β-hydroxybutyrylation to changes in super-enhancers. The method provides a common framework for studying relationships among metabolites, modifications, and regulation, although extending it to other modifications will require further validation.
Highlight · SiTomics links site-specific chromatin modifications to their surrounding proteins and genomic context.
20 · Atlas of Subcellular RNA Localization Revealed by APEX-Seq
Cell · Publication/issue date: 2019
Authors: Furqan M Fazal, Shuo Han, Kevin R Parker, Pornchai Kaewsapsak, Jin Xu, Alistair N Boettiger, Howard Y Chang, Alice Y Ting
An RNA molecule’s subcellular location influences its processing and function, but conventional fractionation methods struggle to resolve complex local environments. APEX-seq directly labels nearby RNA for subsequent sequencing, mapping the human transcriptome across nine subcellular locations and covering different RNA classes and transcript isoforms. The study revealed radial organization of the nuclear transcriptome and suggested that the inner face of the nuclear pore spatially filters mature transcripts for export. Near mitochondria, the authors identified two related mRNA localization pathways associated with distinct transcript sets and machinery within the organelle. By linking RNA sequencing to spatial localization, the work provides an atlas for further exploration. However, proximity enrichment reflects spatial association; additional experiments are needed to establish specific transport or functional mechanisms.
Highlight · APEX-seq links transcript identity to subcellular proximity, revealing how RNA is organized in space.
21 · Split-TurboID enables contact-dependent proximity labeling in cells
Proceedings of the National Academy of Sciences of the United States of America · Publication/issue date: 2020
Authors: Kelvin F Cho, Tess C Branon, Sanjana Rajeev, Tanya Svinkina, Namrata D Udeshi, Themis Thoudam, Chulhwan Kwak, Hyun-Woo Rhee, In-Kyu Lee, Steven A Carr, Alice Y Ting
Conventional proximity labeling can profile local proteomes, but contact sites between two organelles require tighter spatial restriction. Split-TurboID divides the enzyme into two individually inactive fragments that regain labeling activity only when protein interactions or membrane proximity bring them together. Applying the method to endoplasmic reticulum–mitochondria contact sites, the authors identified more than 100 endogenous proteins, including candidates not previously associated with these regions, and validated eight through biochemical fractionation and imaging. By making enzyme activity dependent on contact, the method helps reduce background from labeling across an entire organelle surface. Functional experiments are still needed to determine whether the identified proteins directly participate in contact formation or the exchange of materials at these sites.
Highlight · Split-TurboID uses contact-dependent enzyme reconstitution to label proteins more selectively at organelle interfaces.
22 · Dynamic mapping of proteome trafficking within and between living cells by TransitID
Cell · Publication/issue date: 2023
Authors: Wei Qin, Joleen S Cheah, Charles Xu, James Messing, Brian D Freibaum, Steven Boeynaems, J Paul Taylor, Namrata D Udeshi, Steven A Carr, Alice Y Ting
Knowing where a protein is does not tell us where it came from or where it goes next. TransitID uses two types of proximity labeling systems positioned in source and destination compartments to identify endogenous proteins that pass through both environments, extending mass spectrometry to the study of protein trafficking. The authors examined movement between the cytoplasm and mitochondria or the nucleus, as well as connections between nucleoli and stress granules, finding a link between stress granules and protection of the transcription factor JUN during oxidative stress. The method was also used to identify proteins transferred between macrophages and cancer cells. Its key contribution is to define protein populations by their origin and destination, adding information that static localization maps cannot provide. Specific routes and direct effects still require further validation.
Highlight · By labeling proteins at both their origin and destination, TransitID moves beyond static localization to analyze protein trafficking.
23 · Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging
Science (New York, N.Y.) · Publication/issue date: 2013
Authors: Hyun-Woo Rhee, Peng Zou, Namrata D Udeshi, Jeffrey D Martell, Vamsi K Mootha, Steven A Carr, Alice Y Ting
Microscopy provides spatial information but typically tracks only a few labeled proteins; mass spectrometry can identify large numbers of endogenous proteins but often loses their spatial relationships in living cells. This study combines the strengths of both approaches by targeting a peroxidase to a specific location, labeling nearby proteins, and identifying them by mass spectrometry. Applied to the mitochondrial matrix, the method identified 495 proteins, including 31 not previously associated with mitochondria. Labeling could also distinguish matrix-facing from intermembrane-space-facing regions of inner membrane proteins, and subsequent electron microscopy revised some earlier localization assignments. The work established a way to profile local endogenous proteomes in living cells, opening up microenvironments that are difficult to study using conventional fractionation.
Highlight · Spatially restricted enzymatic labeling preserves information about location in living cells for subsequent protein identification by mass spectrometry.
Authors: Tess C Branon, Justin A Bosch, Ariana D Sanchez, Namrata D Udeshi, Tanya Svinkina, Steven A Carr, Jessica L Feldman, Norbert Perrimon, Alice Y Ting
Early proximity labeling methods often required long labeling periods or reagents with poor permeability or problematic toxicity, limiting studies of short-lived processes and whole organisms. Through directed evolution, the authors developed two biotin ligase variants, TurboID and miniTurbo, with substantially greater labeling efficiency than the earlier BioID system. Under the reported cellular conditions, these variants enabled proximity labeling within shorter time windows using biotin, which is easier to deliver. The study also demonstrated applications in fruit flies and nematodes. The main advance is faster labeling and broader applicability, bringing spatial proteomics to more biological systems. In individual experiments, however, labeling still indicates local proximity: enrichment alone cannot establish direct binding between proteins.
Highlight · TurboID extends efficient biotin-based proximity labeling to shorter time windows and whole organisms.
25 · Directed evolution of APEX2 for electron microscopy and proximity labeling
Nature methods · Publication/issue date: 2015
Authors: Stephanie S Lam, Jeffrey D Martell, Kimberli J Kamer, Thomas J Deerinck, Mark H Ellisman, Vamsi K Mootha, Alice Y Ting
APEX can serve as both an electron microscopy tag and a proximity labeling tool for proteomics in living cells, but limited sensitivity at low expression levels can restrict its use. Through directed evolution, the researchers improved its catalytic efficiency to produce APEX2, which has greater activity in cells. They used it to resolve the submitochondrial localization of MICU1, a regulator of mitochondrial calcium uptake, and to improve enrichment of endogenous proteins associated with mitochondrial and endoplasmic reticulum membranes. Rather than introducing an entirely new localization principle, the study makes an existing tool more useful under low-signal conditions, expanding the range of targets accessible to electron microscopy and local protein profiling. The abstract is brief, and the reported advantages come mainly from these validation systems; they do not directly quantify performance across all cellular compartments.
Highlight · APEX2’s enhanced activity improves the sensitivity of electron microscopy localization and enrichment of nearby proteins within cells.
26 · A fluorophore ligase for site-specific protein labeling inside living cells
Proceedings of the National Academy of Sciences of the United States of America · Publication/issue date: 2010
Authors: Chayasith Uttamapinant, Katharine A White, Hemanta Baruah, Samuel Thompson, Marta Fernández-Suárez, Sujiet Puthenveetil, Alice Y Ting
To reduce the effects of bulky fluorescent protein tags on their targets, PRIME uses a short peptide tag and an engineered ligase to attach small-molecule fluorophores to selected proteins inside living cells. Starting from a natural lipoic acid ligase, the researchers altered substrate recognition so that the enzyme could covalently attach a coumarin probe to the target peptide while maintaining high selectivity over endogenous mammalian proteins in the systems tested. By directing the ligase to different compartments, they further achieved selective labeling of spatially distinct protein populations, such as cell-surface neurexin and nuclear actin. The work shows that labeling specificity can be defined not only by the target sequence but also by the enzyme’s location, offering a smaller-tag alternative for imaging proteins in living cells.
Highlight · PRIME combines short-peptide recognition with subcellular targeting of the enzyme to fluorescently label selected protein populations.
Authors: Jeffrey D Martell, Thomas J Deerinck, Yasemin Sancak, Thomas L Poulos, Vamsi K Mootha, Gina E Sosinsky, Mark H Ellisman, Alice Y Ting
Electron microscopy can reveal nanoscale structures, but many genetically encoded tags are inactive in certain cellular compartments or require illumination, limiting their use. This study developed APEX, a monomeric peroxidase tag that works across multiple cellular compartments and withstands strong fixation conditions, providing a localization signal while preserving ultrastructure. The authors demonstrated imaging of several mammalian organelles and specific proteins. By fusing APEX to the mitochondrial calcium uniporter MCU, they observed electron microscopy contrast at both termini on the matrix side, providing evidence to resolve its disputed topology. The work connects genetically targeted localization with an electron microscopy–visible signal. Because it does not require photoactivation, sample thickness places fewer constraints on labeling.
Highlight · APEX converts genetically targeted localization into electron microscopy contrast and can help determine membrane protein topology.
28 · A monovalent streptavidin with a single femtomolar biotin binding site
Nature methods · Publication/issue date: 2006
Authors: Mark Howarth, Daniel J-F Chinnapen, Kimberly Gerrow, Pieter C Dorrestein, Melanie R Grandy, Neil L Kelleher, Alaa El-Husseini, Alice Y Ting
Streptavidin binds biotin very tightly, but its natural tetramer has multiple binding sites that can crosslink labeled targets, altering their distribution and function. This study retained the overall tetrameric structure while leaving only one functional biotin-binding site, combining monovalent recognition with near-native affinity, dissociation behavior, and thermal stability. In experiments tracking neuroligin-1, the monovalent version stably tracked the target without appreciable crosslinking. By contrast, multivalent wild-type streptavidin caused clustering and disrupted presynaptic contacts. The findings show that the binding valency of an imaging probe is itself an important experimental variable. This tool offers a way to reduce probe-induced clustering in single-particle tracking and biomolecular labeling.
Highlight · Monovalent streptavidin retains strong binding while reducing the target crosslinking caused by multivalent tags.
29 · Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase
Nature methods · Publication/issue date: 2005
Authors: Irwin Chen, Mark Howarth, Weiying Lin, Alice Y Ting
Precise labeling of cell-surface proteins requires distinguishing the target in a complex endogenous environment while accommodating probes with different properties. This study exploits biotin ligase recognition of a short acceptor peptide sequence to introduce reactive chemical groups into the target protein, then attach fluorescent or photoaffinity probes. The authors demonstrate the approach with purified proteins, cell lysates, and EGFR on live cell surfaces, showing that enzymatic selectivity can be combined with subsequent chemical conjugation. The method separates target recognition from probe selection, potentially allowing the same labeling framework to support different biophysical readouts. Because a recognition tag must still be introduced into the target, applications should consider how both this modification and the probe might affect protein localization and function.
Highlight · Combining enzymatic recognition of a short peptide with subsequent chemical conjugation enables cell-surface protein labeling with multiple types of probes.
Authors: Sarah V. Barrass, Tim K. Esser, Nathan J. Mowry, Lukas Eriksson, Jakub Wenz, Laurence T. Seeley, Wyatt A. Curtis, Lindsay A. Baker, Marcel Drabbels, Stephan Rauschenbach, Ulrich J. Lorenz
Soft-landing mass spectrometry could simplify cryo-EM sample preparation, but proteins become dehydrated during deposition, typically leaving only compact conformations observable. In this study, the researchers coat soft-landed samples with amorphous ice, then briefly melt it with a laser to try to restore protein hydration. Treatment produces a particle population spanning multiple partially rehydrated states, some of which approach native conformations. This indicates that dehydration-induced structural changes are at least partly reversible and supports efforts to connect mass spectrometric selection with electron microscopy. The authors outline ways to improve the completeness of rehydration. For now, a key limitation is that only some particles recover; the results do not establish that the entire sample can reliably return to its native state.
Highlight · Brief laser melting allows some soft-landed proteins to recover near-native conformations.
Authors: Yiran Cheng, Bintao He, Qiong Zeng, Fa Zhang, Renmin Han
CryoTriplane uses a triplane neural radiance field and a hybrid-space representation for homogeneous 3D reconstruction from noisy cryo-EM projections. It first establishes a robust overall structure using fixed positional encoding, then adds high-frequency detail through structure-aware encoding. This approach aims to address information-distribution issues in conventional frequency-domain methods and spectral bias in real-space neural networks. The authors compare reconstructions across five simulated and four experimental datasets and assess map quality through downstream atomic modeling. On two specified EMPIAR datasets, sequence coverage improves by 4.6% and 5.5%, respectively, relative to the comparison methods. The work focuses on balancing global consistency with local detail; its reported advantages should be interpreted in the context of the datasets and baselines tested.
Highlight · Hybrid-space encoding helps CryoTriplane balance noise robustness with high-frequency detail in 3D reconstruction.
Quarterly Reviews of Biophysics · Publication/issue date: 2026/01
Authors: Ardan Patwardhan, Peter B. Rosenthal, Christopher J. Russo, Richard Henderson
Starting from the limits imposed by radiation damage, this review estimates how many single-particle images would be needed to achieve different resolutions under ideal imaging conditions, then compares those estimates with published structures in the EMDB. The authors note that image counts for some recent 2.5–3.0 Å structures are already within a factor of three of theoretical expectations. More typical datasets, however, may still require roughly 100 times the theoretical number of images, with similar gaps persisting in high-resolution studies. The review discusses information lost during recording and extraction, along with strategies for narrowing the gap between theory and practice. The authors also emphasize that even excellent structures contain radiation damage, making better use of early, low-dose images an important priority. This provides a fuller perspective on data-collection efficiency than final resolution alone.
Highlight · The gap between theoretical image requirements and actual dataset sizes reveals room to improve information efficiency in single-particle cryo-EM.
33 · Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR
Proceedings of the National Academy of Sciences · Publication/issue date: 2024-11-26
Authors: Jun Xu, Geng Chen, Haoqing Wang, Sheng Cao, Jie Heng, Xavier Deupi, Yang Du, Brian K. Kobilka
Class A GPCRs are small, and without large binding partners such as G proteins, their transmembrane cores are surrounded by detergent micelles, making particle alignment particularly difficult. This study introduces a fusion strategy that connects calcineurin to the receptor at multiple points, providing a more rigid reference outside the membrane. The authors use this approach to determine ligand-free, antagonist-bound, and agonist-bound structures of the β2-adrenergic receptor, showing that adding reliably trackable structural features helps recover information from small membrane proteins. The method's main value lies in expanding the conditions under which structures can be determined without native signaling complexes—not in establishing that all dynamics of the fusion construct match those of the native receptor. Its applicability to other class A GPCRs still requires case-by-case validation.
Highlight · A rigid, multipoint fusion provides an additional reference for aligning small GPCR particles in cryo-EM.
34 · CryoSHIELDs: tunable amphipathic peptide additives for cryo-EM
Preprint · Publication/issue date: 2026-10-08
Authors: Caleigh M. Azumaya, Przemysław Dutka, Matthew C. Johnson, Cheng-Guo Wu, Bryce D. Delgado, Christopher W. Koo, Jeffrey Y. K. Tom, Tik Hang Soong, Sandeep K. Ravala, Alister Burt, Shu Ti, Anna C. Howes, Orlando E. Martinez, Aimin Song, Alexis Rohou, Claudio Ciferri, Stanislau Yatskevich
The air–water interface can damage cryo-EM samples, cause preferred orientation, and reduce particle density. The CryoSHIELDs preprint introduces a set of amphipathic peptide additives with tunable chemical properties, designed to passivate this interface and improve sample condition before vitrification. The authors test 24 systems, including GPCRs, ABC and SLC transporters, soluble complexes, and Fab-containing complexes, and report higher success rates in structure determination. Different additives improve particle distribution, angular sampling, complex stability, or map quality. In some cases, structures can also be obtained at lower protein concentrations. The results support selecting interface-protective additives to suit individual samples, rather than assuming one formulation will work across all systems; performance still needs to be assessed for each target.
Highlight · Tunable amphipathic peptide additives improve cryo-EM performance across multiple sample types by modifying conditions at the air–water interface.
Cryo-electron tomography of tissue requires stable lamellae prepared from thicker material, but serial lift-out cryo-FIB procedures are often complex and prone to lamella instability. This methods paper systematically presents an optimized workflow for multicellular tissues, focusing on lamella mounting, cut spacing, and reliable reattachment to accommodate tissue volumes of different sizes. The authors emphasize that the method can be applied to various biological tissues and integrated with cryogenic correlative light and electron microscopy workflows. It aims to improve structural preservation, reproducibility, and procedural consistency while reducing dependence on operator experience. The paper provides a sample-preparation foundation for in situ structural analysis at the tissue level; the workflow itself should not be confused with the discovery of a specific biological mechanism.
Highlight · Optimizing the mounting and transfer of tissue lamellae helps improve the stability and reproducibility of cryo-ET sample preparation.
NCBE is a sodium-dependent anion exchanger involved in regulating cerebrospinal fluid acid–base balance, but its ion-coupling mechanism has remained unclear. This study resolves multiple cryo-EM states of human NCBE, identifying sodium-, chloride-, and bicarbonate-binding regions, as well as an unexpected potassium site that suggests relatively permissive cation selectivity. The inhibitor DIDS sits above the substrate cavity and stabilizes an outward-facing conformation. Functional experiments and molecular dynamics support the proposed ion coordination and inhibitory effects. Comparing the experimental structures with an inward-facing model predicted by AlphaFold3, the authors propose an elevator-like transport mechanism. Importantly, the inward-facing state is a prediction, so the complete conformational cycle still relies in part on model-based inference.
Highlight · Structures of NCBE in multiple states connect ion coupling and inhibitor recognition with a possible elevator-like transport mechanism.
37 · Structural basis of ligand recognition and gating in mosquito IRs
Nature · Publication/issue date: 2026-10-14
Authors: Li Xu, Xin Wang, Xuebo Quan, Stephen T. Ferguson, Dong Liu, Jiawei Chen, Sai Wang, Simin Liang, Gaoxingyu Huang, Kai Huang, Feng Liu, Xiaojing Pan
Insect ionotropic receptors detect environmental chemical cues, but a structural explanation of how ligand selectivity translates into channel opening has been lacking. This study resolves multiple states of a mosquito IR complex and combines functional measurements with simulations to compare ligand-free, ligand-bound, and open conformations. The results reveal a dynamically adaptable ligand pocket, linking chemical recognition to closure of the extracellular binding domain. This change is then transmitted through linker regions to the transmembrane domain, widening the pore. Together, the evidence supports a framework of mechanical coupling between ligand binding and channel gating. The work offers fundamental structural insight into insect sensory receptors. The vector-control applications mentioned in the abstract remain a future prospect, not a demonstrated practical outcome.
Highlight · Structures in multiple states explain how insect sensory receptors couple chemical recognition to channel gating.
38 · Assembly and architecture of endogenous NMDA receptors in adult cerebral cortex and hippocampus
Cell · Publication/issue date: 2025-03-06
Authors: Ming Zhang, Juan Feng, Chun Xie, Nan Song, Chaozhi Jin, Jian Wang, Qun Zhao, Lihua Zhang, Boshuang Wang, Yidi Sun, Fei Guo, Yang Li, Shujia Zhu
The subunit composition of native NMDA receptors shapes synaptic signaling, but recombinant systems may not fully reflect their actual distribution in the adult brain. Using conformation- and subunit-dependent antibodies, the researchers isolate endogenous receptors from adult rat cortex and hippocampus and determine cryo-EM structures of three major assembly types. Their approximate particle ratio of 9:7:4 suggests substantial co-occurrence of GluN2A and GluN2B subunits in triheteromeric tetramers. These receptors have an asymmetric architecture extending from the extracellular to the transmembrane region, with conformational differences also evident between subtypes. The results reveal the compositional and structural complexity of endogenous receptors, providing a more tissue-relevant basis for understanding subtype function and selective targeting. The particle ratios should still be interpreted in light of the purification and identification conditions.
Highlight · The diverse assemblies of endogenous NMDA receptors in the adult brain reveal structural complexity that a single recombinant subtype cannot capture.
39 · A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion
Cell · Publication/issue date: 2020
Authors: Christina K Kim, Mateo I Sanchez, Paul Hoerbelt, Lief E Fenno, Robert C Malenka, Karl Deisseroth, Alice Y Ting
Identifying and studying cells after a brief burst of neural activity is a key challenge in linking physiological activity to cell type. FLiCRE converts activity-related calcium elevations into stable transcriptional labels, allowing subsequent single-cell sequencing to read both the endogenous transcriptome and a record of past activity. The authors labeled active cells during stimulation of aversive inputs to the mouse nucleus accumbens and identified a specific population activated by long-range excitatory projections. Using the system’s modular output, they then selectively recruited this population and observed aversive behavior. The results connect the recording of transient activity with molecular identification and functional testing. Because the readout integrates activity over time, however, it reflects activity within a defined window rather than individual spikes.
Highlight · FLiCRE links transcriptional records of transient calcium activity to analyses of cell type and behavioral function.
Authors: Wenjing Wang, Craig P Wildes, Tanyaporn Pattarabanjird, Mateo I Sanchez, Gordon F Glober, Gillian A Matthews, Kay M Tye, Alice Y Ting
FLARE is designed to convert neural activity within a researcher-defined time window into gene expression that can be read out later. The system senses both elevated cytosolic calcium and externally delivered light, triggering transcription only when the two coincide and thereby labeling cells active at that time. In cultured neurons, the study demonstrated good discrimination between light and dark conditions and between high and low calcium signals, then used subsequently expressed tools to functionally manipulate the labeled cells. Experiments in the adult mouse cortex also detected transcription associated with light and locomotor activity. By combining temporal control with activity dependence, the design turns transient events into an entry point for subsequent molecular and functional studies. It records activity history within a window rather than directly replacing high-temporal-resolution electrophysiology or calcium imaging.
Highlight · Dual gating by light and calcium gives neurons active within a defined time window a persistent transcriptional label.
De novo design of proteases and membrane proteins (5 papers)
41 · Computational design of metalloproteases
Nature · Publication/issue date: 2026-10-14
Authors: Anqi Chen, Kejia Wu, Hojae Choi, Preetham Venkatesh, Samuel J. Pellock, Nikita Hanikel, Hojeong Shin, Declan Evans, Kieran Didi, Lars L. Schaaf, Cristina Díaz-Perlas, Brian Coventry, Donghyo Kim, Seth M. Woodbury, Pengfei Ji, Shingo Honda, Asim K. Bera, Hannah Nguyen, Alex Kang, Yanqing Wang, Xinting Li, Stacey Gerben, Lemuel Chang, Xiao Yan, Benjamí Oller-Salvia, Anthony A. Hyman, Donald Hilvert, David Baker
Designing enzymes from scratch that can cleave peptide bonds is an important test of whether computational design can tackle reactions with high energy barriers. Using RFdiffusion, the researchers built metalloproteases with a zinc-containing catalytic center and an adjacent substrate-binding pocket. Of 135 designs tested, about 36% cleaved the corresponding peptide substrate at the intended site. The authors report that the best-performing designs substantially accelerated peptide bond hydrolysis, with further optimization yielding additional gains in activity. They also demonstrated selective cleavage of disease-associated proteins and provided proof of concept for using proteases to regulate cell signaling. The results show that computational design can combine catalytic activity with substrate recognition. Therapeutic and biotechnological uses remain potential applications; these experimental results should not be taken to mean that established clinical tools are already available.
Highlight · De novo designed metalloproteases combine relatively high catalytic activity with programmable substrate recognition.
Authors: Hojae Choi, Magnus S. Bauer, Brian Coventry, Preetham Venkatesh, Anqi Chen, Donghyo Kim, Asim K. Bera, Alex Kang, Hannah Nguyen, Shayan Sadre, Justin Decarreau, Emily Joyce, Bhanumathi Shankaran, Tuscan Rock Thompson, Gabriel Greenstein, Kieran Didi, Lars L. Schaaf, Jacob Merle Gershon, Alexander F. Shida, Gyu Rie Lee, Donald Hilvert, Samuel J. Pellock, David Baker
Hydrolyzing stable peptide bonds places stringent demands on the organization of the catalytic center and the positioning of the substrate. The researchers designed proteases from scratch using cysteine as the catalytic nucleophile, obtaining 13 successful designs out of 69 tested. The largest reported rate enhancement was tens of millions of times above background hydrolysis. These proteins have overall folds markedly different from those of natural proteases, while six crystal structures closely matched the design models, supporting the method’s structural accuracy on novel scaffolds. Experimental and computational analyses suggest that inadequate active-site preorganization and substrate positioning still contribute to the activity gap relative to highly active natural enzymes. The authors also demonstrated target cleavage in mammalian cells, showing that the designs can function beyond in vitro reactions. Activity and selectivity nevertheless need to be assessed in each specific system.
Highlight · Designed proteases with nonnatural folds achieve sequence-dependent peptide bond hydrolysis, with structures validating the designs.
43 · De novo designed voltage-gated anion channels suppress neuron firing
Cell · Publication/issue date: 2025/12/24
Authors: Chen Zhou, Huican Li, Jiaxing Wang, Cheng Qian, Hui Xiong, Zhilin Chu, Qiming Shao, Xuan Li, Shijin Sun, Ke Sun, Aiqin Zhu, Jiawei Wang, Xueqin Jin, Fan Yang, Tamer M. Gamal El-Din, Bo Li, Jing Huang, Kun Wu, Peilong Lu
Making a de novo designed membrane protein respond to voltage requires both a conductive pathway and a mechanism coupling environmental changes to conformational movements. The researchers designed voltage-gated anion channels, dVGACs, and observed voltage-dependent anion currents in patch-clamp recordings. Cryo-EM structures closely matched the design models. Together with simulations, they support a role for a charged constriction within the pore in sensing voltage changes and influencing ion selectivity. The authors also report that channel properties can be tuned further and demonstrate suppression of firing in neurons. The work advances computational design beyond static transmembrane structures to dynamic, stimulus-responsive functions, providing an artificial system for studying links between membrane protein conformation and electrical activity. Its range of applications still needs to be assessed in specific cells and experimental conditions.
Highlight · De novo designed anion channels respond to voltage and suppress neuronal firing in experimental systems.
44 · De novo design of dual-topology membrane transporters
Preprint · Publication/issue date: 2025/12/31
Authors: Xi Chen, Xiaofeng Zhou, Jiawei Zhou, Tengyu Xie, Yaning Li, Yuxuan Yan, Jing Huang, Zibo Chen, Dan Ma, Peilong Lu
This preprint explores the de novo construction of membrane proteins that selectively transport small molecules rather than merely form a static pore. The authors designed protein subunits that can adopt opposite membrane topologies, allowing them to assemble into antiparallel dimers. Conformational changes then alternately expose putative substrate-binding regions to either side of the membrane. Experiments in cells and artificial liposomes showed that these proteins facilitate the uptake of specific small-molecule dyes down a concentration gradient, behaving similarly to natural uniporters. Cryo-EM and functional analyses support the designed architecture and proposed transport model. The results demonstrate the potential to build dynamic membrane transport functions from scratch, but the substrate range and generality of the mechanism require further validation. Drug delivery and related uses are presented primarily as future possibilities.
Highlight · Dual-topology design translates the alternating-access concept into measurable transport of small molecules across membranes.
45 · De novo design of transmembrane fluorescence-activating proteins
Nature · Publication/issue date: 2025-04
Authors: Jingyi Zhu, Mingfu Liang, Ke Sun, Yu Wei, Ruiying Guo, Lijing Zhang, Junhui Shi, Dan Ma, Qi Hu, Gaoxingyu Huang, Peilong Lu
Transmembrane proteins must accommodate a lipid environment while forming precise small-molecule-binding pockets, imposing more design constraints than soluble proteins face. This study combined deep learning with energy-based methods to design fluorogen-binding transmembrane proteins from scratch. Experiments showed that the proteins specifically activate ligand fluorescence, bind with mid-nanomolar affinity, and retain activity in live bacteria and eukaryotic membrane fractions. The authors report brightness and quantum yields exceeding those of the enhanced green fluorescent protein used for comparison. Crystal and cryo-EM structures closely matched the design models, validating the intended binding mode. The work shows that protein–small-molecule interactions within membranes can be designed with considerable precision, opening new avenues for membrane-localized imaging and ligand sensing.
Highlight · Structurally validated, designed transmembrane proteins specifically bind fluorogens and activate their fluorescence.
46 · Conservation and specialization of the Ycf2-FtsHi chloroplast protein import motor in green algae
Cell · Publication/issue date: 2024-10-03
Authors: Ke Liang, Xiechao Zhan, Yuxin Li, Yi Yang, Yanqiu Xie, Zeyu Jin, Xiaoyan Xu, Wenwen Zhang, Yang Lu, Sheng Zhang, Yilong Zou, Shan Feng, Jianping Wu, Zhen Yan
Chloroplasts must continually import nuclear-encoded proteins. Whether green algae use the same import motor as land plants is an important question for understanding how this machinery evolved. The researchers isolated the native Ycf2-FtsHi complex from Chlamydomonas and determined its cryo-EM structure, identifying up to 19 subunits, including several components unique to green algae. The heterohexameric AAA+ ATPase motor is tilted relative to the membrane, a configuration that may help it receive precursor proteins from the inner-membrane translocation machinery. In vitro experiments also showed that precursor proteins enhance its ATPase activity. Drawing on the TOC–TIC supercomplex structure, the authors discuss physical and functional links between the channel and motor. The findings reveal both shared operating principles and lineage-specific adaptations, providing a basis for comparing chloroplast protein import across photosynthetic organisms.
Highlight · The green algal import motor retains the core ATPase architecture while incorporating several unique accessory components.
47 · The Structure and Function of the Chloroplast Import Apparatus
Annual Review of Plant Biology · Publication/issue date: 2026-05-20
Authors: Zeyu Jin, Zhen Yan
Many chloroplast proteins are encoded in the nucleus and must be imported through the coordinated action of TOC in the outer membrane, TIC in the inner membrane, and an ATP-driven motor. The composition and operation of this machinery have long been debated, and recent high-resolution structures provide crucial evidence for reassessing its architecture. This review brings together structural, functional, and evolutionary studies to discuss how the channels and motor divide their tasks and assemble, as well as their similarities and differences across photosynthetic organisms. It focuses on how structural biology is revising earlier models and placing previously fragmented functional observations within a more complete molecular framework. Reading it alongside the primary structural papers on green algae and land plants can help distinguish directly supported relationships among components from dynamic steps in import that still need investigation.
Highlight · Recent structures are reshaping our understanding of how TOC, TIC, and the import motor work together.
48 · Structural insights into the chloroplast protein import in land plants
Cell · Publication/issue date: 2024-10-03
Authors: Ke Liang, Zeyu Jin, Xiechao Zhan, Yuxin Li, Qikui Xu, Yanqiu Xie, Yi Yang, Shaojie Wang, Jianping Wu, Zhen Yan
Understanding how precursor proteins pass from the chloroplast inner-membrane channel to the ATPase motor in land plants requires a view of how the two assemble. The researchers determined structures of the Arabidopsis Ycf2-FtsHi and TIC complexes, as well as a larger assembly containing both complexes from pea. The Ycf2-FtsHi structure reveals a heterohexameric AAA+ ATPase module and identifies four previously undercharacterized accessory components involved in assembly and positioning the motor at the membrane surface. The motor sits at an angle close to TIC, spatially aligning the channel exit with the force-generating module. On this basis, the authors propose an arrangement that facilitates efficient precursor transfer. By extending structures of isolated complexes into a coordinated apparatus, the work provides a structural basis for explaining chloroplast import in land plants. Understanding the dynamic process still requires complementary functional evidence.
Highlight · The tilted Ycf2-FtsHi motor closely interfaces with TIC, offering a structural explanation for precursor protein handoff.
49 · Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes
Cell · Publication/issue date: 2022-12-08
Authors: Zeyu Jin, Li Wan, Yuqi Zhang, Xuecheng Li, Yong Cao, Haobin Liu, Shengyao Fan, Du Cao, Zhengmao Wang, Xiaobo Li, Junmin Pan, Meng-Qiu Dong, Jianping Wu, Zhen Yan
Importing nuclear-encoded precursor proteins into chloroplasts requires coordinated translocation across both envelope membranes. This study resolves the cryo-EM structure of the TOC–TIC supercomplex from Chlamydomonas, identifying 14 components and revealing how the complex spans the two membranes. The outer-membrane channel is a hybrid β-barrel assembled from different components, while a potential translocation pathway through the inner membrane is formed by specific transmembrane helices, differing from some conventional models. A rigid scaffold in the intermembrane space connects the two ends, and a hydrophilic cleft along it offers a possible route between the channels. The structure brings composition, assembly, and transport routes into a single framework and provides a reference for comparisons across plants and algae. How precursors actually move through the complex still needs to be tested through dynamic and functional studies.
Highlight · The TOC–TIC supercomplex reveals a continuous structural arrangement connecting the two chloroplast envelope membranes.
50 · Reaction-based cryo-EM resolves the continuous conformational spectrum of CTP synthase catalysis
Proceedings of the National Academy of Sciences · Publication / issue date: 2026-10-20
Authors: Chen-Jun Guo, Yu-Fen Wu, Shu-Ying Guo, You Fu, Wei Wang, Ji-Long Liu
Enzymes undergo continuous changes during catalysis, and locking in a single intermediate in advance can miss this dynamic distribution. Using Drosophila CTP synthase, the researchers sampled an active reaction mixture containing native substrates and allosteric effectors, then applied three-dimensional variability analysis to reconstruct the continuous spectrum of conformations during catalysis. They linked some structural changes to the formation of 4-phosphoryl UTP and observed simultaneous occupancy by ATP and CTP at the reaction endpoint. Comparisons with a nonhydrolyzable ATP analogue indicate that UTP phosphorylation, rather than ATP binding alone, is the key event that shifts the conformational ensemble toward more closed states. By linking chemical progress to conformational distributions, this approach offers a view of enzyme reaction dynamics beyond a single trapped state.
Highlight · Sampling during the reaction links the continuous conformational spectrum of CTPS to actual chemical steps.
GTP promotes CTP synthase catalysis without being consumed, but how its regulatory role is coordinated with the chemical cycle has remained unclear. Combining cryo-EM of active reaction samples with biochemical and mutational analyses, the researchers captured 34 states of Drosophila CTPS at resolutions reaching 2.0 Å. The results support a model in which GTP binds and dissociates once per catalytic cycle, coupled to the production of one CTP molecule. Initial GTP recruitment and stable binding are each constrained by reaction progress in one of the two catalytic domains, synchronizing activity across the domains. The authors call this mechanism effector cycling, emphasizing that the binding and release of an allosteric molecule are themselves part of catalytic regulation, rather than the molecule simply remaining bound at a distant site.
Highlight · Cyclic GTP binding and dissociation coordinate catalysis across the domains of CTPS.
Conventional PROTACs primarily harness intracellular degradation machinery, whereas targeted removal of cell-surface proteins requires a different route. This study introduces GlueTAC, a covalent nanobody-based strategy. Mass spectrometry screening was first used to establish a recognition module capable of proximity-dependent crosslinking to surface antigens; this module was then combined with functional elements that promote cellular uptake and lysosomal sorting. Using PD-L1 as an example, the authors observed target internalization and degradation, demonstrating the potential to turn high-affinity recognition into a signal for clearance. The work broadens the approaches available for membrane protein degradation and provides an experimental system for comparing the roles of noncovalent binding and covalent attachment. The efficiency and specificity reported in the abstract apply to the targets tested; applicability to other membrane proteins still needs to be established case by case.
Highlight · GlueTAC links nanobody recognition to the lysosomal pathway for targeted clearance of cell-surface proteins.
53 · Targeted protein degradation in the transmembrane and extracellular space
Science · Publication / issue date: 2025-11-27
Authors: Rongfeng Zhu, Heng Zhang, Peng R. Chen
Targeted protein degradation has gradually expanded beyond conventional intracellular targets to transmembrane and extracellular proteins, which also play important roles in cell communication and disease progression. This review systematically examines the platforms, mechanisms, applications, and limitations of this emerging field, focusing on how different cellular clearance pathways can bring proteins that are difficult to tackle with conventional PROTACs within reach of degradation. It highlights conceptual advances and clarifies how platforms differ in their requirements for recognition, trafficking, and degradation, rather than treating all strategies as a single technology. The authors also discuss their value for basic research and therapy. Whether these methods can become reliable interventions still depends on factors such as tissue distribution, target accessibility, and the degradation pathways involved.
Highlight · Degradation of transmembrane and extracellular proteins is expanding the target range of TPD while introducing new constraints on trafficking and selectivity.
54 · Development of a random background to understand ligand optimization
Nature · Publication / issue date: 2026-09-16
Authors: Xinyu Xu, Olivier Mailhot, Galen J. Correy, Xi-Ping Huang, Joao M. Braz, Da Shi, Karthik Srinivasan, Kara Zielinski, Yuliia Holota, Yuliia Kuziv, Christos Iliopoulos-Tsoutsouvas, Nathan D. Levinzon, Yagmur U. Doruk, Moira M. Rachman, Morgan E. Diolaiti, Maisie G. V. Stevens, Fangyu Liu, Katie L. Holland, Harald Hübner, Jing Wang, Yujin Wu, Alan Ashworth, Alexandros Makriyannis, Yuqi Zhang, Yurii S. Moroz, Peter Gmeiner, Robert Abel, Aashish Manglik, Allan I. Basbaum, Bryan L. Roth, James S. Fraser, Brian K. Shoichet
Lead optimization often requires synthesizing large numbers of analogues, but without a baseline of random modifications, it is difficult to judge how much deliberate design improves efficiency. This study systematically introduced single-atom changes into 18 lead molecules across six targets, synthesizing 257 compounds in total. The results were unexpected: 11.3% of small random perturbations increased potency at least tenfold, although in vitro pharmacokinetic properties generally worsened. For some compounds, potency gains compensated for reduced exposure and shorter half-lives, yielding stronger effects in vivo. Overall, however, optimization involved trade-offs among different properties. The work provides an initial random baseline for evaluating ligand optimization and shows why judging design success solely by improvements in in vitro activity may overlook major challenges in advancing molecules to in vivo use.
Highlight · Even small random changes can substantially improve potency, but the cost to pharmacokinetic properties may offset the gains.
This issue features 54 papers, focusing on GPCR recognition, conformational dynamics, and signaling selectivity, alongside advances in molecular labeling, spatial omics, and cryo-electron microscopy. Other topics include ion channels, neural activity recording, de novo protein design, chloroplast protein import, CTP synthase allostery, and targeted protein degradation. The studies underscore the importance of integrating structural, dynamic, and functional evidence. New tools and potential therapeutic strategies require further validation, and animal and preclinical findings do not necessarily translate into efficacy or safety advantages in humans.
This issue brings together 54 papers covering GPCR signaling regulation, molecular labeling and spatial omics, cryo-EM methods, ion channels and neural activity, protein design, chloroplast protein import, CTP synthesis, targeted protein degradation, and more.
GPCR Recognition, Dynamics, and Signaling Regulation (17 papers)
01 · An opioid efficacy switch for reversible optical control of analgesia
Cell · Publication/issue date: 2026-10-15
Authors: Luca Posa, Giovanna R. Romano, Xiang Ji, Saif Khan, Antonina L. Nazarova, Sheida Sharghi Moshtaghin, Bruno Paz, Gye Won Han, Anisul Arefin, Alberto J. Gonzalez-Hernandez, Ipsit Srivastava, Saheem A. Zaidi, Kenny Calderon-Godinez, Zixin Zhang, Aaliyah S. Tyson, Mohsen Ranjbar, Kristen E. Pleil, Kanupriya Pande, Vsevolod Katritch, Cornelius Gati, Dirk Trauner, Joshua Levitz
The μ-opioid receptor is a major target for pain relief, but systemic dosing makes it difficult to restrict where a drug acts or separate analgesia from adverse effects. This study developed AM-3, a reversibly photoswitchable agonist that controls receptor signaling by switching between low- and high-efficacy states under illumination. Structures of receptor complexes in the two states reveal distinct binding modes, accompanied by differences in ligand, receptor, and G protein dynamics. In mouse pain models, AM-3 produced reversible, repeatable, light-controlled antinociception. The authors link its reduced adverse effects to limited brain accumulation and the ability to activate it locally with light. The work offers a new tool for studying receptor efficacy and localized analgesia, but the evidence so far comes from animal experiments and does not establish efficacy or safety in humans.
Highlight · Reversible photoswitching changes AM-3’s efficacy at the receptor, enabling localized, repeatable control of analgesia in mice.
DOI: 10.1016/j.cell.2026.09.042
02 · Atomic, structural and molecular insights into GPCR ligand binding and signalling
Nature Reviews Molecular Cell Biology · Publication/issue date: 2026-10-13
Authors: Katarina Nemec, Bálint Mészáros, Georgios Skiniotis, Scott C. Blanchard, M. Madan Babu
Ligand binding and signaling by GPCRs are not a simple on–off switch, but a multilayered process spanning atomic contacts, receptor conformations, and cellular pathways. Moving through increasing levels of complexity, this review brings together recent research on how receptors recognize extracellular signals, undergo ligand-induced conformational changes, and interact with different signaling adaptors and intracellular pathways. It places particular emphasis on context: the behavior of a given receptor depends on its molecular partners, regulatory state, and disease environment, so static structures explain only part of the picture. The authors also discuss how these insights could guide more selective interventions. The review’s value lies in its multiscale framework linking structure to function, helping readers understand why findings at different experimental levels are connected but not directly interchangeable.
Highlight · Understanding GPCR pharmacology requires a unified view of ligand contacts, conformational changes, and cellular signaling.
DOI: 10.1038/s41580-026-01030-7
03 · The Role of Intrinsically Disordered Domains in Regulating G Protein-Coupled Receptor Signaling
Journal of the American Chemical Society · Publication/issue date: 2026-01-14
Authors: Jun Xu, Ruoyi Qiu, Alexander M. Garces, Harald Hübner, Xinyu Xu, Chengyu Hao, Dorothee Weikert, Peter Gmeiner, Michael T. Lerch, Brian K. Kobilka
Signaling by the α2A-adrenergic receptor is shaped not only by its transmembrane core but potentially also by its flexible, disordered regions. Using multiple single-molecule FRET labeling configurations to track conformational changes in real time, the researchers found a high energy barrier to the outward movement of TM6 required for activation, while the partially disordered extracellular loop ECL2 showed ligand-specific dynamics. The long third intracellular loop, ICL3, adopted a relatively compact conformation close to the membrane in the basal state and negatively modulated coupling to signaling transducers through an allosteric mechanism. Its multiple functional substates were also influenced by kinase phosphorylation and drug efficacy. These findings recast disordered loops as dynamic regulatory modules rather than passive linkers, helping explain how the same receptor can produce distinct signaling profiles in response to different ligands.
Highlight · Dynamic conformations of disordered GPCR loops can regulate ligand efficacy and coupling to signaling proteins.
DOI: 10.1021/jacs.5c17796
04 · Single-molecule analysis of ligand efficacy in β2AR–G-protein activation
Nature · Publication/issue date: 2017-07
Authors: G. Glenn Gregorio, Matthieu Masureel, Daniel Hilger, Daniel S. Terry, Manuel Juette, Hong Zhao, Zhou Zhou, Jose Manuel Perez-Aguilar, Maria Hauge, Signe Mathiasen, Jonathan A. Javitch, Harel Weinstein, Brian K. Kobilka, Scott C. Blanchard
Differences in GPCR ligand efficacy may arise from distinct dynamics during receptor and G protein activation. This study used single-molecule FRET to monitor TM6 movement in the β2-adrenergic receptor, comparing ligands of different efficacies in the presence and absence of Gs. Partial and full agonists modulated TM6 in different ways, affecting both the rate of GDP-bound receptor–Gs complex formation and the efficiency of subsequent nucleotide exchange. The experiments also captured transient nucleotide-bound states not adequately represented by existing static structures. By tracing the allosteric connection between the ligand-binding pocket and the G protein’s nucleotide-binding pocket through these dynamic processes, the work suggests that drug efficacy depends not just on an end-state conformation, but also on the rates and pathways of transitions between states.
Highlight · Ligands produce different levels of Gs activation by altering receptor motions and nucleotide exchange kinetics.
DOI: 10.1038/nature22354
05 · Ligand efficacy modulates conformational dynamics of the µ-opioid receptor
Nature · Publication/issue date: 2024-05
Authors: Jiawei Zhao, Matthias Elgeti, Evan S. O’Brien, Cecília P. Sár, Amal EI Daibani, Jie Heng, Xiaoou Sun, Elizabeth White, Tao Che, Wayne L. Hubbell, Brian K. Kobilka, Chunlai Chen
The μ-opioid receptor is a major target for analgesic drugs, and understanding how ligands produce different intrinsic efficacies requires a view of conformational dynamics rather than a single structure. Combining double electron–electron resonance with single-molecule FRET, the researchers identified multiple states of the receptor’s cytoplasmic side that interconvert on different timescales. A preactivated conformation could bind G proteins, whereas a fully activated conformation substantially reduced GDP affinity in the ternary complex, showing that binding and productive activation are distinct steps. The authors also compared how β-arrestin-1 and Gi interact with the receptor core, finding lower affinity and weaker specificity for β-arrestin-1. These results link ligand-specific conformational distributions to downstream efficacy, providing a dynamic basis for analyzing opioid receptor signaling selectivity.
Highlight · Preactivated and fully activated states of the μ-opioid receptor play distinct roles in G protein binding and GDP release.
DOI: 10.1038/s41586-024-07295-2
06 · Ligand-dependent G protein dynamics underlying opioid efficacy
Nature Structural & Molecular Biology · Publication/issue date: 2026-10-13
Authors: Jonathan C. Deutsch, Daniel Hilger, John Janetzko, Charles M. Schroeder, Steven Chu, Brian K. Kobilka, Rabindra V. Shivnaraine
Following receptor activation, the G protein’s α-helical domain must open to expose the nucleotide-binding site and allow nucleotide exchange. This study used single-molecule FRET to directly observe Gi coupled to the μ-opioid receptor, comparing domain motions across different ligands and nucleotide states. The results show that ligands bound at a distant site on the receptor can influence G protein dynamics, with higher-efficacy agonists more readily driving the protein into open states with low nucleotide affinity. The experiments also captured transient intermediates during nucleotide exchange. By extending the study of drug action beyond the receptor to its coupling partner, the work shows that allosteric modulation of the G protein’s conformational ensemble is another mechanism that encodes opioid ligand efficacy, rather than a wholly passive consequence of receptor changes.
Highlight · Differences in opioid ligand efficacy can propagate to Gi domain motions and nucleotide exchange.
DOI: 10.1038/s41594-026-01893-6
07 · Structure and dynamics determine G protein coupling specificity at a class A GPCR
Science Advances · Publication/issue date: 2025-03-19
Authors: Marina Casiraghi, Haoqing Wang, Patrick C. Brennan, Chris Habrian, Harald Hübner, Maximilian F. Schmidt, Luis Maul, Biswaranjan Pani, Sherif M. F. M. Bahriz, Bing Xu, Nico Staffen, Tufa E. Assafa, Bohan Chen, Elizabeth White, Roger K. Sunahara, Asuka Inoue, Yang K. Xiang, Robert J. Lefkowitz, Ehud Y. Isacoff, Nathaniel Nucci, Peter Gmeiner, Michael T. Lerch, Brian K. Kobilka
The β2-adrenergic receptor generally favors Gs and couples more weakly to Gi, but the mere presence or absence of a complex cannot explain this selectivity. After identifying the Gi-biased agonist LM189, the researchers combined structural and biophysical evidence to compare the receptor states required for coupling to different G proteins. Their results indicate that specific conformations of intracellular loop ICL2 and transmembrane helix TM6 work together to distinguish Gs from Gi, with receptor dynamics also contributing to this preference. The work thus links G protein selectivity to variable conformational features within the receptor, rather than treating it as a fixed property of its sequence. This provides a mechanistic basis for understanding biased agonists and highlights the need to consider both structural states and functional measurements when assessing signaling selectivity.
Highlight · Conformational differences in ICL2 and TM6 jointly influence the β2 receptor’s preference for coupling to different G proteins.
DOI: 10.1126/sciadv.adq3971
08 · Discovery of a functionally selective serotonin receptor (5-HT1AR) agonist for the treatment of pain
Science Advances · Publication/issue date: 2025-06-18
Authors: Annika Ullrich, Johannes Schneider, João M. Braz, Eduard Neu, Nico Staffen, Markus Stanek, Jana Bláhová, Tamsanqa Hove, Tamara Albert, Anni Allikalt, Stefan Löber, Karnika Bhardwaj, Sian Rodriguez-Rosado, Elissa Fink, Tim Rasmussen, Harald Hübner, Asuka Inoue, Brian K. Shoichet, Allan I. Basbaum, Bettina Böttcher, Dorothee Weikert, Peter Gmeiner
The 5-HT1A receptor participates in pain regulation, but how to harness signaling bias to achieve a desirable pharmacological profile remains an important research question. Starting from a compound library, the authors identified the bitopic agonist ST171, which primarily activated Gi/o, showed no Gs activation and weak β-arrestin recruitment, and had effects in acute and chronic pain models. Cryo-EM comparisons of the ST171 complex with Gi and Gs complexes of the conventional agonist befiradol showed that the ligands occupy different peripheral sites and are associated with distinct receptor conformations. Molecular dynamics simulations helped explain these differences. The study connects binding modes, signaling selectivity, and analgesic effects in experimental models, but these remain mechanistic and preclinical findings; they do not directly establish efficacy or a safety advantage in humans.
Highlight · Distinct peripheral binding modes and receptor conformations provide a structural explanation for signaling bias among 5-HT1A agonists.
DOI: 10.1126/sciadv.adv9267
09 · Mechanism and function of GPR3 regulated by a negative allosteric modulator
Nature Communications · Publication/issue date: 2025-08-27
Authors: Geng Chen, Jana Bláhová, Nico Staffen, Harald Hübner, Nadja Nunhöfer, Chen Qiu, Peter Gmeiner, Dorothee Weikert, Yang Du, Jun Xu
GPCR dimerization can alter how drugs act, and GPR3 offers an example of dimer-specific modulation. The study found that GPR3 forms constitutive homodimers in living cells, with the inhibitor AF64394 acting on this state as a negative allosteric modulator. Cryo-EM structures of the inhibitor-bound dimer and its Gs complex place the ligand at the transmembrane dimer interface. The authors propose that the ligand prevents the receptors from dissociating upon engagement with Gs and constrains the relevant transmembrane regions in an inactive-like intermediate conformation, thereby reducing Gs coupling. Together, the structural and functional findings show that receptor oligomerization can confer pharmacological selectivity and underscore the need to consider receptor–receptor interactions when studying allosteric inhibition.
Highlight · The GPR3 dimer interface offers a route to negative allosteric modulation distinct from conventional ligand-binding pockets.
DOI: 10.1038/s41467-025-63422-1
10 · A TM7 conformational switch governs GPCR transducer selectivity
Preprint · Publication/issue date: 2026/04/21
Authors: Guodong He, Maik Pankonin, Guojing Yu, Haiyan Xu, Huan Li, Maximilian Salomon, Hossein Batebi, Xue Guo, Shuhao Zhang, Fang Kong, Li Zhan, Xiaoou Sun, Qingqing Ma, Lan Hua, Sheng Ding, Chuangye Yan, Xin Chen, Zhaobing Gao, Peter W. Hildebrand, Xiangyu Liu
This preprint examines how the μ-opioid receptor selects among different signaling partners. The authors identified an agonist with pronounced G protein bias and determined cryo-EM structures of its receptor–Gi and receptor–GRK2 complexes. Combining these structures with functional and dynamics data, they propose a stepwise gating model. In this model, conformational changes in TM7 act as a final checkpoint that can influence subsequent β-arrestin recruitment: even after completing some activation steps, the receptor need not proceed along every signaling pathway. The work frames biased pharmacology as selection within a multistep process, rather than simply a difference between active conformations. The evidence remains limited to the structural and experimental systems reported in this preprint and does not establish that the agonist offers a clinical safety advantage.
Highlight · TM7 dynamics may act as a final checkpoint in determining which signaling partners a GPCR engages.
DOI: 10.65215/LTSpreprints.2026.04.21.000162
11 · Structural basis for Y2 receptor-mediated neuropeptide Y and peptide YY signaling
Structure · Publication/issue date: 2023-01-05
Authors: Hyunook Kang, Chaehee Park, Yeol Kyo Choi, Jungnam Bae, Sohee Kwon, Jinuk Kim, Chulwon Choi, Chaok Seok, Wonpil Im, Hee-Jung Choi
Receptors in the neuropeptide Y family play distinct roles in appetite and mood regulation, and even small changes in a ligand can alter receptor selectivity. The study determined cryo-EM structures of the Y2 receptor–Gi complex bound to either PYY(3–36) or NPY. Together with cell-based experiments and simulations, these structures explain why PYY lacking its first two amino acids can still act effectively on Y2 but cannot similarly activate Y1. Comparing downstream pathways, the authors further found that Y2 favors G protein signaling upon activation, whereas Y1 did not show the same preference between G protein and β-arrestin signaling. The work links differences in ligand recognition to signaling bias, suggesting that even receptors within the same family cannot simply be assumed to share the same models of ligand binding and function.
Highlight · Both its ligand recognition features and its preference for G protein signaling distinguish Y2 from the related Y1 receptor.
DOI: 10.1016/j.str.2022.11.010
12 · Structural basis of neuropeptide Y signaling through Y1 receptor
Nature Communications · Publication/issue date: 2022-02-14
Authors: Chaehee Park, Jinuk Kim, Seung-Bum Ko, Yeol Kyo Choi, Hyeongseop Jeong, Hyeonuk Woo, Hyunook Kang, Injin Bang, Sang Ah Kim, Tae-Young Yoon, Chaok Seok, Wonpil Im, Hee-Jung Choi
NPY participates in many physiological processes, including feeding and anxiety, but a complete structural account of its interactions with the Y1 receptor had been lacking. The study resolved the NPY–Y1–Gi1 complex, showing that the peptide’s extended C terminus enters the receptor’s transmembrane core, with its amidated terminal residue sitting at the bottom of the binding pocket. Meanwhile, the helical and N-terminal regions of NPY contact the receptor’s extracellular loops, further contributing to high-affinity binding. Structural analysis and mutagenesis together support this coordinated, multiregion recognition model. The findings help explain why different parts of the intact peptide can influence activity and provide a basis for comparing selectivity across Y1 and other NPY receptors, rather than attributing all pharmacological properties to a single local contact.
Highlight · NPY binding within the transmembrane pocket and its contacts with extracellular loops jointly support Y1 receptor recognition and activation.
DOI: 10.1038/s41467-022-28510-6
13 · Structure-based discovery of nonopioid analgesics acting through the α2A-adrenergic receptor
Science · Publication/issue date: 2022-09-30
Authors: Elissa A. Fink, Jun Xu, Harald Hübner, Joao M. Braz, Philipp Seemann, Charlotte Avet, Veronica Craik, Dorothee Weikert, Maximilian F. Schmidt, Chase M. Webb, Nataliya A. Tolmachova, Yurii S. Moroz, Xi-Ping Huang, Chakrapani Kalyanaraman, Stefan Gahbauer, Geng Chen, Zheng Liu, Matthew P. Jacobson, John J. Irwin, Michel Bouvier, Yang Du, Brian K. Shoichet, Allan I. Basbaum, Peter Gmeiner
The α2A-adrenergic receptor is a potential target for nonopioid pain relief, but existing drugs often cause sedation. The researchers screened more than 300 million virtual molecules by docking to find new scaffolds distinct from those of commonly used drugs. They identified a set of active ligands, many of which showed partial agonism and a preference for Gi and Go signaling. Experimentally determined complex structures for two agonists supported the docking predictions and provided a basis for further optimization. Several initial hits and analogues produced target-dependent analgesia in multiple in vivo pain models without corresponding signs of sedation. The work demonstrates how large-scale structure-based screening can be combined with experimental validation and shows that ligands acting at the same receptor can differ in their pharmacological effects. These findings remain preclinical, however, and do not directly establish a therapeutic advantage in humans.
Highlight · New α2A agonists identified through structure-based screening suggest that analgesia may be separable from sedation in animal models.
DOI: 10.1126/science.abn7065
14 · G protein-coupled receptors in neurodegenerative diseases and psychiatric disorders
Signal Transduction and Targeted Therapy · Publication/issue date: 2023-05-03
Authors: Thian-Sze Wong, Guangzhi Li, Shiliang Li, Wei Gao, Geng Chen, Shiyi Gan, Manzhan Zhang, Honglin Li, Song Wu, Yang Du
Neurodegenerative diseases and psychiatric disorders have complex, heterogeneous causes, and a single molecular change rarely explains the full range of symptoms. This review takes a GPCR-centered approach, summarizing the roles of different receptors in these conditions and how studies of receptor mechanisms and structures can create new opportunities for drug development. Focusing on emerging targets and recent advances in drug research, the authors place receptor-level insights in the broader context of multifactorial disease. The article offers an overview of targets and research directions; it does not imply that every receptor discussed has been validated to the same extent as a therapeutic target. Its value lies in connecting biological questions in neuropsychiatric disease with receptor systems that can be modulated, while acknowledging the evidence still needed to translate basic mechanisms into effective interventions.
Highlight · GPCRs offer multiple avenues for intervention in neuropsychiatric disease, but their value as targets must be assessed in light of disease heterogeneity.
DOI: 10.1038/s41392-023-01427-2
15 · Cryo-EM structure of monomeric CXCL12-bound CXCR4 in the active state
Cell Reports · Publication/issue date: 2024-08-27
Authors: Yezhou Liu, Aijun Liu, Xinyu Li, Qiwen Liao, Weijia Zhang, Lizhe Zhu, Richard D. Ye
CXCL12–CXCR4 signaling contributes to hematopoietic cell localization and tumor-related processes, but the agonist-bound active structure had not been fully characterized. The study obtained a high-resolution cryo-EM structure of the CXCL12–CXCR4–Gi complex, showing one-to-one ligand–receptor binding consistent with the two-site model of chemokine recognition. The ligand’s N terminus enters the transmembrane pocket and makes contacts within the receptor that help drive activation. Structural, functional, and computational analyses together explain how these interactions connect to receptor conformational changes and G protein activation. By adding an active-state view to existing antagonist-bound structures, the work provides a foundation for understanding the receptor’s different pharmacological states, but does not directly establish a new clinical intervention.
Highlight · The CXCL12-bound active-state structure fills in the mechanistic picture of CXCR4 signaling, from ligand recognition to G protein activation.
DOI: 10.1016/j.celrep.2024.114578
16 · Photo-cross-linking-assisted deorphanization deciphers GPR50–L-LEN pairing in metabolism
Nature Chemical Biology · Publication/issue date: 2026-07
Authors: Rui Wu, Na Li, Zhihui Wen, Ya Wang, Shitian Li, Hao Zhou, Yuehong Huang, Xiao Xie, Xiaoyun Zhao, Xiaohui Wang, Shaoxin Tao, Guoliang Chai, Tongfei A. Wang, Zhen Li, Peng R. Chen, Miao Jing
Many GPCRs still lack known endogenous ligands, limiting studies of their physiological functions and pharmacology. The authors developed a photo-cross-linking-assisted receptor deorphanization platform that captures ligands at the recognition interface to identify them in native biological samples. They tested its performance on several known receptor–ligand pairs. The study further identifies the neuropeptide L-LEN as a proposed endogenous ligand for GPR50, with evidence that it regulates cellular activity through this receptor and downstream Gi signaling. In mice, this pairing is linked to brain–adipose communication, energy expenditure, and thermogenesis; loss of relevant components increases the likelihood of entering a torpor-like state under stress. The work provides both a tool for ligand discovery and clues to metabolic regulation, although its applicability across biological settings requires further investigation.
Highlight · Photo-cross-linking-assisted capture identifies a candidate endogenous ligand for GPR50 and links the receptor to body temperature and energy regulation.
DOI: 10.1038/s41589-025-02098-6
17 · Synthetic GPCRs for programmable sensing and control of cell behaviour
Nature · Publication/issue date: 2025
Authors: Nicholas A Kalogriopoulos, Reika Tei, Yuqi Yan, Peter M Klein, Matthew Ravalin, Bo Cai, Ivan Soltesz, Yulong Li, Alice Y Ting
Synthetic receptors can translate specific antigens into cellular responses, but existing systems face limitations in the forms of antigen they recognize, the outputs they produce, and their amenability to drug control. The study introduces PAGER synthetic GPCRs, in which antigen binding relieves receptor inhibition, enabling a subsequent response to a drug and thus linking recognition to a controllable signaling output. The authors demonstrate responses to more than a dozen soluble and cell-surface antigens, with different receptor scaffolds driving gene expression, fluorescent readouts, or endogenous G protein activity. Applications include cell migration, differentiation, antibody secretion, and modulation of neural activity in brain slices. The work illustrates the versatility of modular receptor engineering, but validation across these settings does not mean that all corresponding therapeutic applications are ready for use; each still requires evaluation in its specific cellular context.
Highlight · PAGER combines antigen recognition, drug control, and multiple GPCR outputs in a programmable platform for cellular sensing.
DOI: 10.1038/s41586-024-08282-3
Molecular labeling, imaging, and spatial omics (12 papers)
18 · Recent developments of fluorescent proteins and biosensors driven by cross-disciplinary insights
Nature Methods · Publication/issue date: 2026-10-16
Authors: Anneliese M. M. Gest, Sohum Mehta, Jin Zhang
Advances in fluorescent proteins and biosensors increasingly depend on the interplay between chemistry, physics, protein engineering, and screening technologies. This review surveys recent progress in fluorescent protein engineering, focusing on design goals such as improving photostability and shifting emission to longer wavelengths. Using mScarlet as a case study, it illustrates how insights from different fields can translate into better performance. The article also summarizes developments in fluorescent sensors for screening, quantitative imaging, spatial resolution, and multiplexed detection, considering how probes and measurement methods together expand what can be observed. Finally, the authors discuss directions for next-generation fluorescent tools. The article is a broad review of existing technologies and design approaches, rather than a performance report on a single new probe.
Highlight · Progress in fluorescent tools comes from coordinated advances in molecular design, screening, and imaging methods.
DOI: 10.1038/s41592-026-03268-7
19 · Linking chromatin acylation mark-defined proteome and genome in living cells
Cell · Publication/issue date: 2023-03-02
Authors: Fangfei Qin, Boyuan Li, Hui Wang, Sihui Ma, Jiaofeng Li, Shanglin Liu, Linghao Kong, Huangtao Zheng, Rongfeng Zhu, Yu Han, Mingdong Yang, Kai Li, Xiong Ji, Peng R. Chen
Histone modifications are linked to metabolic state and can alter chromatin interactions and gene regulation, but directly connecting a modification at a specific site to its local environment is challenging. This study introduces SiTomics, a multiomics strategy with single-site resolution that profiles the proteins and genomic regions associated with specific chromatin acylation marks in living cells. Comparing crotonylation and β-hydroxybutyrylation following short-chain fatty acid stimulation, the authors found distinct association profiles and identified GLYR1 as an interacting protein that helps regulate H3K56cr localization within gene bodies. The results also linked some effects of β-hydroxybutyrylation to changes in super-enhancers. The method provides a common framework for studying relationships among metabolites, modifications, and regulation, although extending it to other modifications will require further validation.
Highlight · SiTomics links site-specific chromatin modifications to their surrounding proteins and genomic context.
DOI: 10.1016/j.cell.2023.02.007
20 · Atlas of Subcellular RNA Localization Revealed by APEX-Seq
Cell · Publication/issue date: 2019
Authors: Furqan M Fazal, Shuo Han, Kevin R Parker, Pornchai Kaewsapsak, Jin Xu, Alistair N Boettiger, Howard Y Chang, Alice Y Ting
An RNA molecule’s subcellular location influences its processing and function, but conventional fractionation methods struggle to resolve complex local environments. APEX-seq directly labels nearby RNA for subsequent sequencing, mapping the human transcriptome across nine subcellular locations and covering different RNA classes and transcript isoforms. The study revealed radial organization of the nuclear transcriptome and suggested that the inner face of the nuclear pore spatially filters mature transcripts for export. Near mitochondria, the authors identified two related mRNA localization pathways associated with distinct transcript sets and machinery within the organelle. By linking RNA sequencing to spatial localization, the work provides an atlas for further exploration. However, proximity enrichment reflects spatial association; additional experiments are needed to establish specific transport or functional mechanisms.
Highlight · APEX-seq links transcript identity to subcellular proximity, revealing how RNA is organized in space.
DOI: 10.1016/j.cell.2019.05.027
21 · Split-TurboID enables contact-dependent proximity labeling in cells
Proceedings of the National Academy of Sciences of the United States of America · Publication/issue date: 2020
Authors: Kelvin F Cho, Tess C Branon, Sanjana Rajeev, Tanya Svinkina, Namrata D Udeshi, Themis Thoudam, Chulhwan Kwak, Hyun-Woo Rhee, In-Kyu Lee, Steven A Carr, Alice Y Ting
Conventional proximity labeling can profile local proteomes, but contact sites between two organelles require tighter spatial restriction. Split-TurboID divides the enzyme into two individually inactive fragments that regain labeling activity only when protein interactions or membrane proximity bring them together. Applying the method to endoplasmic reticulum–mitochondria contact sites, the authors identified more than 100 endogenous proteins, including candidates not previously associated with these regions, and validated eight through biochemical fractionation and imaging. By making enzyme activity dependent on contact, the method helps reduce background from labeling across an entire organelle surface. Functional experiments are still needed to determine whether the identified proteins directly participate in contact formation or the exchange of materials at these sites.
Highlight · Split-TurboID uses contact-dependent enzyme reconstitution to label proteins more selectively at organelle interfaces.
DOI: 10.1073/pnas.1919528117
22 · Dynamic mapping of proteome trafficking within and between living cells by TransitID
Cell · Publication/issue date: 2023
Authors: Wei Qin, Joleen S Cheah, Charles Xu, James Messing, Brian D Freibaum, Steven Boeynaems, J Paul Taylor, Namrata D Udeshi, Steven A Carr, Alice Y Ting
Knowing where a protein is does not tell us where it came from or where it goes next. TransitID uses two types of proximity labeling systems positioned in source and destination compartments to identify endogenous proteins that pass through both environments, extending mass spectrometry to the study of protein trafficking. The authors examined movement between the cytoplasm and mitochondria or the nucleus, as well as connections between nucleoli and stress granules, finding a link between stress granules and protection of the transcription factor JUN during oxidative stress. The method was also used to identify proteins transferred between macrophages and cancer cells. Its key contribution is to define protein populations by their origin and destination, adding information that static localization maps cannot provide. Specific routes and direct effects still require further validation.
Highlight · By labeling proteins at both their origin and destination, TransitID moves beyond static localization to analyze protein trafficking.
DOI: 10.1016/j.cell.2023.05.044
23 · Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging
Science (New York, N.Y.) · Publication/issue date: 2013
Authors: Hyun-Woo Rhee, Peng Zou, Namrata D Udeshi, Jeffrey D Martell, Vamsi K Mootha, Steven A Carr, Alice Y Ting
Microscopy provides spatial information but typically tracks only a few labeled proteins; mass spectrometry can identify large numbers of endogenous proteins but often loses their spatial relationships in living cells. This study combines the strengths of both approaches by targeting a peroxidase to a specific location, labeling nearby proteins, and identifying them by mass spectrometry. Applied to the mitochondrial matrix, the method identified 495 proteins, including 31 not previously associated with mitochondria. Labeling could also distinguish matrix-facing from intermembrane-space-facing regions of inner membrane proteins, and subsequent electron microscopy revised some earlier localization assignments. The work established a way to profile local endogenous proteomes in living cells, opening up microenvironments that are difficult to study using conventional fractionation.
Highlight · Spatially restricted enzymatic labeling preserves information about location in living cells for subsequent protein identification by mass spectrometry.
DOI: 10.1126/science.1230593
24 · Efficient proximity labeling in living cells and organisms with TurboID
Nature biotechnology · Publication/issue date: 2018
Authors: Tess C Branon, Justin A Bosch, Ariana D Sanchez, Namrata D Udeshi, Tanya Svinkina, Steven A Carr, Jessica L Feldman, Norbert Perrimon, Alice Y Ting
Early proximity labeling methods often required long labeling periods or reagents with poor permeability or problematic toxicity, limiting studies of short-lived processes and whole organisms. Through directed evolution, the authors developed two biotin ligase variants, TurboID and miniTurbo, with substantially greater labeling efficiency than the earlier BioID system. Under the reported cellular conditions, these variants enabled proximity labeling within shorter time windows using biotin, which is easier to deliver. The study also demonstrated applications in fruit flies and nematodes. The main advance is faster labeling and broader applicability, bringing spatial proteomics to more biological systems. In individual experiments, however, labeling still indicates local proximity: enrichment alone cannot establish direct binding between proteins.
Highlight · TurboID extends efficient biotin-based proximity labeling to shorter time windows and whole organisms.
DOI: 10.1038/nbt.4201
25 · Directed evolution of APEX2 for electron microscopy and proximity labeling
Nature methods · Publication/issue date: 2015
Authors: Stephanie S Lam, Jeffrey D Martell, Kimberli J Kamer, Thomas J Deerinck, Mark H Ellisman, Vamsi K Mootha, Alice Y Ting
APEX can serve as both an electron microscopy tag and a proximity labeling tool for proteomics in living cells, but limited sensitivity at low expression levels can restrict its use. Through directed evolution, the researchers improved its catalytic efficiency to produce APEX2, which has greater activity in cells. They used it to resolve the submitochondrial localization of MICU1, a regulator of mitochondrial calcium uptake, and to improve enrichment of endogenous proteins associated with mitochondrial and endoplasmic reticulum membranes. Rather than introducing an entirely new localization principle, the study makes an existing tool more useful under low-signal conditions, expanding the range of targets accessible to electron microscopy and local protein profiling. The abstract is brief, and the reported advantages come mainly from these validation systems; they do not directly quantify performance across all cellular compartments.
Highlight · APEX2’s enhanced activity improves the sensitivity of electron microscopy localization and enrichment of nearby proteins within cells.
DOI: 10.1038/nmeth.3179
26 · A fluorophore ligase for site-specific protein labeling inside living cells
Proceedings of the National Academy of Sciences of the United States of America · Publication/issue date: 2010
Authors: Chayasith Uttamapinant, Katharine A White, Hemanta Baruah, Samuel Thompson, Marta Fernández-Suárez, Sujiet Puthenveetil, Alice Y Ting
To reduce the effects of bulky fluorescent protein tags on their targets, PRIME uses a short peptide tag and an engineered ligase to attach small-molecule fluorophores to selected proteins inside living cells. Starting from a natural lipoic acid ligase, the researchers altered substrate recognition so that the enzyme could covalently attach a coumarin probe to the target peptide while maintaining high selectivity over endogenous mammalian proteins in the systems tested. By directing the ligase to different compartments, they further achieved selective labeling of spatially distinct protein populations, such as cell-surface neurexin and nuclear actin. The work shows that labeling specificity can be defined not only by the target sequence but also by the enzyme’s location, offering a smaller-tag alternative for imaging proteins in living cells.
Highlight · PRIME combines short-peptide recognition with subcellular targeting of the enzyme to fluorescently label selected protein populations.
DOI: 10.1073/pnas.0914067107
27 · Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy
Nature biotechnology · Publication/issue date: 2012
Authors: Jeffrey D Martell, Thomas J Deerinck, Yasemin Sancak, Thomas L Poulos, Vamsi K Mootha, Gina E Sosinsky, Mark H Ellisman, Alice Y Ting
Electron microscopy can reveal nanoscale structures, but many genetically encoded tags are inactive in certain cellular compartments or require illumination, limiting their use. This study developed APEX, a monomeric peroxidase tag that works across multiple cellular compartments and withstands strong fixation conditions, providing a localization signal while preserving ultrastructure. The authors demonstrated imaging of several mammalian organelles and specific proteins. By fusing APEX to the mitochondrial calcium uniporter MCU, they observed electron microscopy contrast at both termini on the matrix side, providing evidence to resolve its disputed topology. The work connects genetically targeted localization with an electron microscopy–visible signal. Because it does not require photoactivation, sample thickness places fewer constraints on labeling.
Highlight · APEX converts genetically targeted localization into electron microscopy contrast and can help determine membrane protein topology.
DOI: 10.1038/nbt.2375
28 · A monovalent streptavidin with a single femtomolar biotin binding site
Nature methods · Publication/issue date: 2006
Authors: Mark Howarth, Daniel J-F Chinnapen, Kimberly Gerrow, Pieter C Dorrestein, Melanie R Grandy, Neil L Kelleher, Alaa El-Husseini, Alice Y Ting
Streptavidin binds biotin very tightly, but its natural tetramer has multiple binding sites that can crosslink labeled targets, altering their distribution and function. This study retained the overall tetrameric structure while leaving only one functional biotin-binding site, combining monovalent recognition with near-native affinity, dissociation behavior, and thermal stability. In experiments tracking neuroligin-1, the monovalent version stably tracked the target without appreciable crosslinking. By contrast, multivalent wild-type streptavidin caused clustering and disrupted presynaptic contacts. The findings show that the binding valency of an imaging probe is itself an important experimental variable. This tool offers a way to reduce probe-induced clustering in single-particle tracking and biomolecular labeling.
Highlight · Monovalent streptavidin retains strong binding while reducing the target crosslinking caused by multivalent tags.
DOI: 10.1038/nmeth861
29 · Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase
Nature methods · Publication/issue date: 2005
Authors: Irwin Chen, Mark Howarth, Weiying Lin, Alice Y Ting
Precise labeling of cell-surface proteins requires distinguishing the target in a complex endogenous environment while accommodating probes with different properties. This study exploits biotin ligase recognition of a short acceptor peptide sequence to introduce reactive chemical groups into the target protein, then attach fluorescent or photoaffinity probes. The authors demonstrate the approach with purified proteins, cell lysates, and EGFR on live cell surfaces, showing that enzymatic selectivity can be combined with subsequent chemical conjugation. The method separates target recognition from probe selection, potentially allowing the same labeling framework to support different biophysical readouts. Because a recognition tag must still be introduced into the target, applications should consider how both this modification and the probe might affect protein localization and function.
Highlight · Combining enzymatic recognition of a short peptide with subsequent chemical conjugation enables cell-surface protein labeling with multiple types of probes.
DOI: 10.1038/nmeth735
Cryo-EM Sample Preparation and Reconstruction (6 papers)
30 · Cryo-EM sample preparation with soft-landing and laser flash melting
Nature Communications · Publication/issue date: 2026-09-15
Authors: Sarah V. Barrass, Tim K. Esser, Nathan J. Mowry, Lukas Eriksson, Jakub Wenz, Laurence T. Seeley, Wyatt A. Curtis, Lindsay A. Baker, Marcel Drabbels, Stephan Rauschenbach, Ulrich J. Lorenz
Soft-landing mass spectrometry could simplify cryo-EM sample preparation, but proteins become dehydrated during deposition, typically leaving only compact conformations observable. In this study, the researchers coat soft-landed samples with amorphous ice, then briefly melt it with a laser to try to restore protein hydration. Treatment produces a particle population spanning multiple partially rehydrated states, some of which approach native conformations. This indicates that dehydration-induced structural changes are at least partly reversible and supports efforts to connect mass spectrometric selection with electron microscopy. The authors outline ways to improve the completeness of rehydration. For now, a key limitation is that only some particles recover; the results do not establish that the entire sample can reliably return to its native state.
Highlight · Brief laser melting allows some soft-landed proteins to recover near-native conformations.
DOI: 10.1038/s41467-026-77723-6
31 · CryoTriplane: Triplane-based Neural Radiance Field Representation for High-resolution Cryo-EM 3D Homogeneous Reconstruction
Bioinformatics · Publication/issue date: 2026-10-13
Authors: Yiran Cheng, Bintao He, Qiong Zeng, Fa Zhang, Renmin Han
CryoTriplane uses a triplane neural radiance field and a hybrid-space representation for homogeneous 3D reconstruction from noisy cryo-EM projections. It first establishes a robust overall structure using fixed positional encoding, then adds high-frequency detail through structure-aware encoding. This approach aims to address information-distribution issues in conventional frequency-domain methods and spectral bias in real-space neural networks. The authors compare reconstructions across five simulated and four experimental datasets and assess map quality through downstream atomic modeling. On two specified EMPIAR datasets, sequence coverage improves by 4.6% and 5.5%, respectively, relative to the comparison methods. The work focuses on balancing global consistency with local detail; its reported advantages should be interpreted in the context of the datasets and baselines tested.
Highlight · Hybrid-space encoding helps CryoTriplane balance noise robustness with high-frequency detail in 3D reconstruction.
DOI: 10.1093/bioinformatics/btag754
32 · Single-particle cryoEM: Theory and practice
Quarterly Reviews of Biophysics · Publication/issue date: 2026/01
Authors: Ardan Patwardhan, Peter B. Rosenthal, Christopher J. Russo, Richard Henderson
Starting from the limits imposed by radiation damage, this review estimates how many single-particle images would be needed to achieve different resolutions under ideal imaging conditions, then compares those estimates with published structures in the EMDB. The authors note that image counts for some recent 2.5–3.0 Å structures are already within a factor of three of theoretical expectations. More typical datasets, however, may still require roughly 100 times the theoretical number of images, with similar gaps persisting in high-resolution studies. The review discusses information lost during recording and extraction, along with strategies for narrowing the gap between theory and practice. The authors also emphasize that even excellent structures contain radiation damage, making better use of early, low-dose images an important priority. This provides a fuller perspective on data-collection efficiency than final resolution alone.
Highlight · The gap between theoretical image requirements and actual dataset sizes reveals room to improve information efficiency in single-particle cryo-EM.
DOI: 10.1017/S0033583526100249
33 · Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR
Proceedings of the National Academy of Sciences · Publication/issue date: 2024-11-26
Authors: Jun Xu, Geng Chen, Haoqing Wang, Sheng Cao, Jie Heng, Xavier Deupi, Yang Du, Brian K. Kobilka
Class A GPCRs are small, and without large binding partners such as G proteins, their transmembrane cores are surrounded by detergent micelles, making particle alignment particularly difficult. This study introduces a fusion strategy that connects calcineurin to the receptor at multiple points, providing a more rigid reference outside the membrane. The authors use this approach to determine ligand-free, antagonist-bound, and agonist-bound structures of the β2-adrenergic receptor, showing that adding reliably trackable structural features helps recover information from small membrane proteins. The method's main value lies in expanding the conditions under which structures can be determined without native signaling complexes—not in establishing that all dynamics of the fusion construct match those of the native receptor. Its applicability to other class A GPCRs still requires case-by-case validation.
Highlight · A rigid, multipoint fusion provides an additional reference for aligning small GPCR particles in cryo-EM.
DOI: 10.1073/pnas.2414544121
34 · CryoSHIELDs: tunable amphipathic peptide additives for cryo-EM
Preprint · Publication/issue date: 2026-10-08
Authors: Caleigh M. Azumaya, Przemysław Dutka, Matthew C. Johnson, Cheng-Guo Wu, Bryce D. Delgado, Christopher W. Koo, Jeffrey Y. K. Tom, Tik Hang Soong, Sandeep K. Ravala, Alister Burt, Shu Ti, Anna C. Howes, Orlando E. Martinez, Aimin Song, Alexis Rohou, Claudio Ciferri, Stanislau Yatskevich
The air–water interface can damage cryo-EM samples, cause preferred orientation, and reduce particle density. The CryoSHIELDs preprint introduces a set of amphipathic peptide additives with tunable chemical properties, designed to passivate this interface and improve sample condition before vitrification. The authors test 24 systems, including GPCRs, ABC and SLC transporters, soluble complexes, and Fab-containing complexes, and report higher success rates in structure determination. Different additives improve particle distribution, angular sampling, complex stability, or map quality. In some cases, structures can also be obtained at lower protein concentrations. The results support selecting interface-protective additives to suit individual samples, rather than assuming one formulation will work across all systems; performance still needs to be assessed for each target.
Highlight · Tunable amphipathic peptide additives improve cryo-EM performance across multiple sample types by modifying conditions at the air–water interface.
DOI: 10.64898/2026.10.01.755993
35 · Step-by-step procedure for an optimized serial lift-out cryo-focused ion beam milling technique in tissue analysis
Biophysics Reports · Publication/issue date: 2025
Authors: Xiaoyu Tang, Jiaming Liu, Jingtao Zheng, Huanhuan Huang, Zihang Yu, Ying Liu, Peng Wang, Meijing Li
Cryo-electron tomography of tissue requires stable lamellae prepared from thicker material, but serial lift-out cryo-FIB procedures are often complex and prone to lamella instability. This methods paper systematically presents an optimized workflow for multicellular tissues, focusing on lamella mounting, cut spacing, and reliable reattachment to accommodate tissue volumes of different sizes. The authors emphasize that the method can be applied to various biological tissues and integrated with cryogenic correlative light and electron microscopy workflows. It aims to improve structural preservation, reproducibility, and procedural consistency while reducing dependence on operator experience. The paper provides a sample-preparation foundation for in situ structural analysis at the tissue level; the workflow itself should not be confused with the discovery of a specific biological mechanism.
Highlight · Optimizing the mounting and transfer of tissue lamellae helps improve the stability and reproducibility of cryo-ET sample preparation.
DOI: 10.52601/bpr.2025.250039
Ion Channels, Transport, and Neural Activity Recording (5 papers)
36 · Substrate coupling and inhibition in human Na+-dependent Cl−/HCO3− exchanger NCBE (SLC4A10)
Nature Communications · Publication/issue date: 2026-10-01
Authors: Peixin Sun, Xinmin Wang, Yang Huang, Jinlun Kylian Zhang, Xurui Shen, Anqi Dong, Shiliang Wang, Xiaojun Guo, Guoming Shi, Jing Ding, Yudan Chi, Hanting Yang
NCBE is a sodium-dependent anion exchanger involved in regulating cerebrospinal fluid acid–base balance, but its ion-coupling mechanism has remained unclear. This study resolves multiple cryo-EM states of human NCBE, identifying sodium-, chloride-, and bicarbonate-binding regions, as well as an unexpected potassium site that suggests relatively permissive cation selectivity. The inhibitor DIDS sits above the substrate cavity and stabilizes an outward-facing conformation. Functional experiments and molecular dynamics support the proposed ion coordination and inhibitory effects. Comparing the experimental structures with an inward-facing model predicted by AlphaFold3, the authors propose an elevator-like transport mechanism. Importantly, the inward-facing state is a prediction, so the complete conformational cycle still relies in part on model-based inference.
Highlight · Structures of NCBE in multiple states connect ion coupling and inhibitor recognition with a possible elevator-like transport mechanism.
DOI: 10.1038/s41467-026-77504-1
37 · Structural basis of ligand recognition and gating in mosquito IRs
Nature · Publication/issue date: 2026-10-14
Authors: Li Xu, Xin Wang, Xuebo Quan, Stephen T. Ferguson, Dong Liu, Jiawei Chen, Sai Wang, Simin Liang, Gaoxingyu Huang, Kai Huang, Feng Liu, Xiaojing Pan
Insect ionotropic receptors detect environmental chemical cues, but a structural explanation of how ligand selectivity translates into channel opening has been lacking. This study resolves multiple states of a mosquito IR complex and combines functional measurements with simulations to compare ligand-free, ligand-bound, and open conformations. The results reveal a dynamically adaptable ligand pocket, linking chemical recognition to closure of the extracellular binding domain. This change is then transmitted through linker regions to the transmembrane domain, widening the pore. Together, the evidence supports a framework of mechanical coupling between ligand binding and channel gating. The work offers fundamental structural insight into insect sensory receptors. The vector-control applications mentioned in the abstract remain a future prospect, not a demonstrated practical outcome.
Highlight · Structures in multiple states explain how insect sensory receptors couple chemical recognition to channel gating.
DOI: 10.1038/s41586-026-11239-3
38 · Assembly and architecture of endogenous NMDA receptors in adult cerebral cortex and hippocampus
Cell · Publication/issue date: 2025-03-06
Authors: Ming Zhang, Juan Feng, Chun Xie, Nan Song, Chaozhi Jin, Jian Wang, Qun Zhao, Lihua Zhang, Boshuang Wang, Yidi Sun, Fei Guo, Yang Li, Shujia Zhu
The subunit composition of native NMDA receptors shapes synaptic signaling, but recombinant systems may not fully reflect their actual distribution in the adult brain. Using conformation- and subunit-dependent antibodies, the researchers isolate endogenous receptors from adult rat cortex and hippocampus and determine cryo-EM structures of three major assembly types. Their approximate particle ratio of 9:7:4 suggests substantial co-occurrence of GluN2A and GluN2B subunits in triheteromeric tetramers. These receptors have an asymmetric architecture extending from the extracellular to the transmembrane region, with conformational differences also evident between subtypes. The results reveal the compositional and structural complexity of endogenous receptors, providing a more tissue-relevant basis for understanding subtype function and selective targeting. The particle ratios should still be interpreted in light of the purification and identification conditions.
Highlight · The diverse assemblies of endogenous NMDA receptors in the adult brain reveal structural complexity that a single recombinant subtype cannot capture.
DOI: 10.1016/j.cell.2025.01.004
39 · A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving Aversion
Cell · Publication/issue date: 2020
Authors: Christina K Kim, Mateo I Sanchez, Paul Hoerbelt, Lief E Fenno, Robert C Malenka, Karl Deisseroth, Alice Y Ting
Identifying and studying cells after a brief burst of neural activity is a key challenge in linking physiological activity to cell type. FLiCRE converts activity-related calcium elevations into stable transcriptional labels, allowing subsequent single-cell sequencing to read both the endogenous transcriptome and a record of past activity. The authors labeled active cells during stimulation of aversive inputs to the mouse nucleus accumbens and identified a specific population activated by long-range excitatory projections. Using the system’s modular output, they then selectively recruited this population and observed aversive behavior. The results connect the recording of transient activity with molecular identification and functional testing. Because the readout integrates activity over time, however, it reflects activity within a defined window rather than individual spikes.
Highlight · FLiCRE links transcriptional records of transient calcium activity to analyses of cell type and behavioral function.
DOI: 10.1016/j.cell.2020.11.015
40 · A light- and calcium-gated transcription factor for imaging and manipulating activated neurons
Nature biotechnology · Publication/issue date: 2017
Authors: Wenjing Wang, Craig P Wildes, Tanyaporn Pattarabanjird, Mateo I Sanchez, Gordon F Glober, Gillian A Matthews, Kay M Tye, Alice Y Ting
FLARE is designed to convert neural activity within a researcher-defined time window into gene expression that can be read out later. The system senses both elevated cytosolic calcium and externally delivered light, triggering transcription only when the two coincide and thereby labeling cells active at that time. In cultured neurons, the study demonstrated good discrimination between light and dark conditions and between high and low calcium signals, then used subsequently expressed tools to functionally manipulate the labeled cells. Experiments in the adult mouse cortex also detected transcription associated with light and locomotor activity. By combining temporal control with activity dependence, the design turns transient events into an entry point for subsequent molecular and functional studies. It records activity history within a window rather than directly replacing high-temporal-resolution electrophysiology or calcium imaging.
Highlight · Dual gating by light and calcium gives neurons active within a defined time window a persistent transcriptional label.
DOI: 10.1038/nbt.3909
De novo design of proteases and membrane proteins (5 papers)
41 · Computational design of metalloproteases
Nature · Publication/issue date: 2026-10-14
Authors: Anqi Chen, Kejia Wu, Hojae Choi, Preetham Venkatesh, Samuel J. Pellock, Nikita Hanikel, Hojeong Shin, Declan Evans, Kieran Didi, Lars L. Schaaf, Cristina Díaz-Perlas, Brian Coventry, Donghyo Kim, Seth M. Woodbury, Pengfei Ji, Shingo Honda, Asim K. Bera, Hannah Nguyen, Alex Kang, Yanqing Wang, Xinting Li, Stacey Gerben, Lemuel Chang, Xiao Yan, Benjamí Oller-Salvia, Anthony A. Hyman, Donald Hilvert, David Baker
Designing enzymes from scratch that can cleave peptide bonds is an important test of whether computational design can tackle reactions with high energy barriers. Using RFdiffusion, the researchers built metalloproteases with a zinc-containing catalytic center and an adjacent substrate-binding pocket. Of 135 designs tested, about 36% cleaved the corresponding peptide substrate at the intended site. The authors report that the best-performing designs substantially accelerated peptide bond hydrolysis, with further optimization yielding additional gains in activity. They also demonstrated selective cleavage of disease-associated proteins and provided proof of concept for using proteases to regulate cell signaling. The results show that computational design can combine catalytic activity with substrate recognition. Therapeutic and biotechnological uses remain potential applications; these experimental results should not be taken to mean that established clinical tools are already available.
Highlight · De novo designed metalloproteases combine relatively high catalytic activity with programmable substrate recognition.
DOI: 10.1038/s41586-026-11167-2
42 · De novo design of cysteine proteases
Nature · Publication/issue date: 2026-10-14
Authors: Hojae Choi, Magnus S. Bauer, Brian Coventry, Preetham Venkatesh, Anqi Chen, Donghyo Kim, Asim K. Bera, Alex Kang, Hannah Nguyen, Shayan Sadre, Justin Decarreau, Emily Joyce, Bhanumathi Shankaran, Tuscan Rock Thompson, Gabriel Greenstein, Kieran Didi, Lars L. Schaaf, Jacob Merle Gershon, Alexander F. Shida, Gyu Rie Lee, Donald Hilvert, Samuel J. Pellock, David Baker
Hydrolyzing stable peptide bonds places stringent demands on the organization of the catalytic center and the positioning of the substrate. The researchers designed proteases from scratch using cysteine as the catalytic nucleophile, obtaining 13 successful designs out of 69 tested. The largest reported rate enhancement was tens of millions of times above background hydrolysis. These proteins have overall folds markedly different from those of natural proteases, while six crystal structures closely matched the design models, supporting the method’s structural accuracy on novel scaffolds. Experimental and computational analyses suggest that inadequate active-site preorganization and substrate positioning still contribute to the activity gap relative to highly active natural enzymes. The authors also demonstrated target cleavage in mammalian cells, showing that the designs can function beyond in vitro reactions. Activity and selectivity nevertheless need to be assessed in each specific system.
Highlight · Designed proteases with nonnatural folds achieve sequence-dependent peptide bond hydrolysis, with structures validating the designs.
DOI: 10.1038/s41586-026-11143-w
43 · De novo designed voltage-gated anion channels suppress neuron firing
Cell · Publication/issue date: 2025/12/24
Authors: Chen Zhou, Huican Li, Jiaxing Wang, Cheng Qian, Hui Xiong, Zhilin Chu, Qiming Shao, Xuan Li, Shijin Sun, Ke Sun, Aiqin Zhu, Jiawei Wang, Xueqin Jin, Fan Yang, Tamer M. Gamal El-Din, Bo Li, Jing Huang, Kun Wu, Peilong Lu
Making a de novo designed membrane protein respond to voltage requires both a conductive pathway and a mechanism coupling environmental changes to conformational movements. The researchers designed voltage-gated anion channels, dVGACs, and observed voltage-dependent anion currents in patch-clamp recordings. Cryo-EM structures closely matched the design models. Together with simulations, they support a role for a charged constriction within the pore in sensing voltage changes and influencing ion selectivity. The authors also report that channel properties can be tuned further and demonstrate suppression of firing in neurons. The work advances computational design beyond static transmembrane structures to dynamic, stimulus-responsive functions, providing an artificial system for studying links between membrane protein conformation and electrical activity. Its range of applications still needs to be assessed in specific cells and experimental conditions.
Highlight · De novo designed anion channels respond to voltage and suppress neuronal firing in experimental systems.
DOI: 10.1016/j.cell.2025.09.023
44 · De novo design of dual-topology membrane transporters
Preprint · Publication/issue date: 2025/12/31
Authors: Xi Chen, Xiaofeng Zhou, Jiawei Zhou, Tengyu Xie, Yaning Li, Yuxuan Yan, Jing Huang, Zibo Chen, Dan Ma, Peilong Lu
This preprint explores the de novo construction of membrane proteins that selectively transport small molecules rather than merely form a static pore. The authors designed protein subunits that can adopt opposite membrane topologies, allowing them to assemble into antiparallel dimers. Conformational changes then alternately expose putative substrate-binding regions to either side of the membrane. Experiments in cells and artificial liposomes showed that these proteins facilitate the uptake of specific small-molecule dyes down a concentration gradient, behaving similarly to natural uniporters. Cryo-EM and functional analyses support the designed architecture and proposed transport model. The results demonstrate the potential to build dynamic membrane transport functions from scratch, but the substrate range and generality of the mechanism require further validation. Drug delivery and related uses are presented primarily as future possibilities.
Highlight · Dual-topology design translates the alternating-access concept into measurable transport of small molecules across membranes.
DOI: 10.65215/LTSpreprints.2025.12.30.000074
45 · De novo design of transmembrane fluorescence-activating proteins
Nature · Publication/issue date: 2025-04
Authors: Jingyi Zhu, Mingfu Liang, Ke Sun, Yu Wei, Ruiying Guo, Lijing Zhang, Junhui Shi, Dan Ma, Qi Hu, Gaoxingyu Huang, Peilong Lu
Transmembrane proteins must accommodate a lipid environment while forming precise small-molecule-binding pockets, imposing more design constraints than soluble proteins face. This study combined deep learning with energy-based methods to design fluorogen-binding transmembrane proteins from scratch. Experiments showed that the proteins specifically activate ligand fluorescence, bind with mid-nanomolar affinity, and retain activity in live bacteria and eukaryotic membrane fractions. The authors report brightness and quantum yields exceeding those of the enhanced green fluorescent protein used for comparison. Crystal and cryo-EM structures closely matched the design models, validating the intended binding mode. The work shows that protein–small-molecule interactions within membranes can be designed with considerable precision, opening new avenues for membrane-localized imaging and ligand sensing.
Highlight · Structurally validated, designed transmembrane proteins specifically bind fluorogens and activate their fluorescence.
DOI: 10.1038/s41586-025-08598-8
Chloroplast protein import (4 papers)
46 · Conservation and specialization of the Ycf2-FtsHi chloroplast protein import motor in green algae
Cell · Publication/issue date: 2024-10-03
Authors: Ke Liang, Xiechao Zhan, Yuxin Li, Yi Yang, Yanqiu Xie, Zeyu Jin, Xiaoyan Xu, Wenwen Zhang, Yang Lu, Sheng Zhang, Yilong Zou, Shan Feng, Jianping Wu, Zhen Yan
Chloroplasts must continually import nuclear-encoded proteins. Whether green algae use the same import motor as land plants is an important question for understanding how this machinery evolved. The researchers isolated the native Ycf2-FtsHi complex from Chlamydomonas and determined its cryo-EM structure, identifying up to 19 subunits, including several components unique to green algae. The heterohexameric AAA+ ATPase motor is tilted relative to the membrane, a configuration that may help it receive precursor proteins from the inner-membrane translocation machinery. In vitro experiments also showed that precursor proteins enhance its ATPase activity. Drawing on the TOC–TIC supercomplex structure, the authors discuss physical and functional links between the channel and motor. The findings reveal both shared operating principles and lineage-specific adaptations, providing a basis for comparing chloroplast protein import across photosynthetic organisms.
Highlight · The green algal import motor retains the core ATPase architecture while incorporating several unique accessory components.
DOI: 10.1016/j.cell.2024.08.002
47 · The Structure and Function of the Chloroplast Import Apparatus
Annual Review of Plant Biology · Publication/issue date: 2026-05-20
Authors: Zeyu Jin, Zhen Yan
Many chloroplast proteins are encoded in the nucleus and must be imported through the coordinated action of TOC in the outer membrane, TIC in the inner membrane, and an ATP-driven motor. The composition and operation of this machinery have long been debated, and recent high-resolution structures provide crucial evidence for reassessing its architecture. This review brings together structural, functional, and evolutionary studies to discuss how the channels and motor divide their tasks and assemble, as well as their similarities and differences across photosynthetic organisms. It focuses on how structural biology is revising earlier models and placing previously fragmented functional observations within a more complete molecular framework. Reading it alongside the primary structural papers on green algae and land plants can help distinguish directly supported relationships among components from dynamic steps in import that still need investigation.
Highlight · Recent structures are reshaping our understanding of how TOC, TIC, and the import motor work together.
DOI: 10.1146/annurev-arplant-062325-090826
48 · Structural insights into the chloroplast protein import in land plants
Cell · Publication/issue date: 2024-10-03
Authors: Ke Liang, Zeyu Jin, Xiechao Zhan, Yuxin Li, Qikui Xu, Yanqiu Xie, Yi Yang, Shaojie Wang, Jianping Wu, Zhen Yan
Understanding how precursor proteins pass from the chloroplast inner-membrane channel to the ATPase motor in land plants requires a view of how the two assemble. The researchers determined structures of the Arabidopsis Ycf2-FtsHi and TIC complexes, as well as a larger assembly containing both complexes from pea. The Ycf2-FtsHi structure reveals a heterohexameric AAA+ ATPase module and identifies four previously undercharacterized accessory components involved in assembly and positioning the motor at the membrane surface. The motor sits at an angle close to TIC, spatially aligning the channel exit with the force-generating module. On this basis, the authors propose an arrangement that facilitates efficient precursor transfer. By extending structures of isolated complexes into a coordinated apparatus, the work provides a structural basis for explaining chloroplast import in land plants. Understanding the dynamic process still requires complementary functional evidence.
Highlight · The tilted Ycf2-FtsHi motor closely interfaces with TIC, offering a structural explanation for precursor protein handoff.
DOI: 10.1016/j.cell.2024.08.003
49 · Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes
Cell · Publication/issue date: 2022-12-08
Authors: Zeyu Jin, Li Wan, Yuqi Zhang, Xuecheng Li, Yong Cao, Haobin Liu, Shengyao Fan, Du Cao, Zhengmao Wang, Xiaobo Li, Junmin Pan, Meng-Qiu Dong, Jianping Wu, Zhen Yan
Importing nuclear-encoded precursor proteins into chloroplasts requires coordinated translocation across both envelope membranes. This study resolves the cryo-EM structure of the TOC–TIC supercomplex from Chlamydomonas, identifying 14 components and revealing how the complex spans the two membranes. The outer-membrane channel is a hybrid β-barrel assembled from different components, while a potential translocation pathway through the inner membrane is formed by specific transmembrane helices, differing from some conventional models. A rigid scaffold in the intermembrane space connects the two ends, and a hydrophilic cleft along it offers a possible route between the channels. The structure brings composition, assembly, and transport routes into a single framework and provides a reference for comparisons across plants and algae. How precursors actually move through the complex still needs to be tested through dynamic and functional studies.
Highlight · The TOC–TIC supercomplex reveals a continuous structural arrangement connecting the two chloroplast envelope membranes.
DOI: 10.1016/j.cell.2022.10.030
CTP synthase catalysis and allostery (2 papers)
50 · Reaction-based cryo-EM resolves the continuous conformational spectrum of CTP synthase catalysis
Proceedings of the National Academy of Sciences · Publication / issue date: 2026-10-20
Authors: Chen-Jun Guo, Yu-Fen Wu, Shu-Ying Guo, You Fu, Wei Wang, Ji-Long Liu
Enzymes undergo continuous changes during catalysis, and locking in a single intermediate in advance can miss this dynamic distribution. Using Drosophila CTP synthase, the researchers sampled an active reaction mixture containing native substrates and allosteric effectors, then applied three-dimensional variability analysis to reconstruct the continuous spectrum of conformations during catalysis. They linked some structural changes to the formation of 4-phosphoryl UTP and observed simultaneous occupancy by ATP and CTP at the reaction endpoint. Comparisons with a nonhydrolyzable ATP analogue indicate that UTP phosphorylation, rather than ATP binding alone, is the key event that shifts the conformational ensemble toward more closed states. By linking chemical progress to conformational distributions, this approach offers a view of enzyme reaction dynamics beyond a single trapped state.
Highlight · Sampling during the reaction links the continuous conformational spectrum of CTPS to actual chemical steps.
DOI: 10.1073/pnas.2620271123
51 · GTP orchestrates CTP synthase via an allosteric effector–cycling mechanism
Proceedings of the National Academy of Sciences · Publication / issue date: 2026-10-20
Authors: Chen-Jun Guo, Yu-Fen Wu, Shu-Ying Guo, Jia-Li Lu, Liang Xu, You Fu, Xian Zhou, Jiale Zhong, Zherong Zhang, Ji-Long Liu
GTP promotes CTP synthase catalysis without being consumed, but how its regulatory role is coordinated with the chemical cycle has remained unclear. Combining cryo-EM of active reaction samples with biochemical and mutational analyses, the researchers captured 34 states of Drosophila CTPS at resolutions reaching 2.0 Å. The results support a model in which GTP binds and dissociates once per catalytic cycle, coupled to the production of one CTP molecule. Initial GTP recruitment and stable binding are each constrained by reaction progress in one of the two catalytic domains, synchronizing activity across the domains. The authors call this mechanism effector cycling, emphasizing that the binding and release of an allosteric molecule are themselves part of catalytic regulation, rather than the molecule simply remaining bound at a distant site.
Highlight · Cyclic GTP binding and dissociation coordinate catalysis across the domains of CTPS.
DOI: 10.1073/pnas.2620272123
Degradation of cell-surface and extracellular proteins (2 papers)
52 · Covalently Engineered Nanobody Chimeras for Targeted Membrane Protein Degradation
Journal of the American Chemical Society · Publication / issue date: 2021-10-13
Authors: Heng Zhang, Yu Han, Yuanfan Yang, Feng Lin, Kexin Li, Linghao Kong, Hongxiang Liu, Yongjun Dang, Jian Lin, Peng R. Chen
Conventional PROTACs primarily harness intracellular degradation machinery, whereas targeted removal of cell-surface proteins requires a different route. This study introduces GlueTAC, a covalent nanobody-based strategy. Mass spectrometry screening was first used to establish a recognition module capable of proximity-dependent crosslinking to surface antigens; this module was then combined with functional elements that promote cellular uptake and lysosomal sorting. Using PD-L1 as an example, the authors observed target internalization and degradation, demonstrating the potential to turn high-affinity recognition into a signal for clearance. The work broadens the approaches available for membrane protein degradation and provides an experimental system for comparing the roles of noncovalent binding and covalent attachment. The efficiency and specificity reported in the abstract apply to the targets tested; applicability to other membrane proteins still needs to be established case by case.
Highlight · GlueTAC links nanobody recognition to the lysosomal pathway for targeted clearance of cell-surface proteins.
DOI: 10.1021/jacs.1c08521
53 · Targeted protein degradation in the transmembrane and extracellular space
Science · Publication / issue date: 2025-11-27
Authors: Rongfeng Zhu, Heng Zhang, Peng R. Chen
Targeted protein degradation has gradually expanded beyond conventional intracellular targets to transmembrane and extracellular proteins, which also play important roles in cell communication and disease progression. This review systematically examines the platforms, mechanisms, applications, and limitations of this emerging field, focusing on how different cellular clearance pathways can bring proteins that are difficult to tackle with conventional PROTACs within reach of degradation. It highlights conceptual advances and clarifies how platforms differ in their requirements for recognition, trafficking, and degradation, rather than treating all strategies as a single technology. The authors also discuss their value for basic research and therapy. Whether these methods can become reliable interventions still depends on factors such as tissue distribution, target accessibility, and the degradation pathways involved.
Highlight · Degradation of transmembrane and extracellular proteins is expanding the target range of TPD while introducing new constraints on trafficking and selectivity.
DOI: 10.1126/science.adx5094
Other papers (1 paper)
54 · Development of a random background to understand ligand optimization
Nature · Publication / issue date: 2026-09-16
Authors: Xinyu Xu, Olivier Mailhot, Galen J. Correy, Xi-Ping Huang, Joao M. Braz, Da Shi, Karthik Srinivasan, Kara Zielinski, Yuliia Holota, Yuliia Kuziv, Christos Iliopoulos-Tsoutsouvas, Nathan D. Levinzon, Yagmur U. Doruk, Moira M. Rachman, Morgan E. Diolaiti, Maisie G. V. Stevens, Fangyu Liu, Katie L. Holland, Harald Hübner, Jing Wang, Yujin Wu, Alan Ashworth, Alexandros Makriyannis, Yuqi Zhang, Yurii S. Moroz, Peter Gmeiner, Robert Abel, Aashish Manglik, Allan I. Basbaum, Bryan L. Roth, James S. Fraser, Brian K. Shoichet
Lead optimization often requires synthesizing large numbers of analogues, but without a baseline of random modifications, it is difficult to judge how much deliberate design improves efficiency. This study systematically introduced single-atom changes into 18 lead molecules across six targets, synthesizing 257 compounds in total. The results were unexpected: 11.3% of small random perturbations increased potency at least tenfold, although in vitro pharmacokinetic properties generally worsened. For some compounds, potency gains compensated for reduced exposure and shorter half-lives, yielding stronger effects in vivo. Overall, however, optimization involved trade-offs among different properties. The work provides an initial random baseline for evaluating ligand optimization and shows why judging design success solely by improvements in in vitro activity may overlook major challenges in advancing molecules to in vivo use.
Highlight · Even small random changes can substantially improve potency, but the cost to pharmacokinetic properties may offset the gains.
DOI: 10.1038/s41586-026-11013-5