Cryo-electron tomography (cryo-ET) enables the determination of high-resolution three-dimensional structures of macromolecular complexes within cells in a near-physiological state, providing crucial structural insights into fundamental life processes. Cryo-ET has achieved landmark successes in single-cell models. However, many critical biological processes do not occur in isolated cells but emerge from intercellular coordination within tissues. Furthermore, many research subjects, including neural tissues, tumor biopsies, plant tissues, and clinical pathological samples, cannot be obtained through single-cell culture and must be directly dissected from organisms or tissue blocks. Advancing cryo-ET from single-cell to tissue-level applications is therefore crucial for capturing the full complexity of biological activities in their native context. A major technical bottleneck for tissue cryo-ET lies in the preparation of sufficiently thin (<300 nm) lamellae from vitrified tissue specimens. Although high-pressure freezing can vitrify tissues up to 200 µm thick, these samples are far too thick for direct transmission electron microscopy imaging. Among the available thinning methods, cryo-focused ion beam (cryo-FIB) milling has emerged as the most promising approach, as it avoids the mechanical artifacts inherent to cryo-ultramicrotomy. However, conventional on-the-grid cryo-FIB milling is inefficient for thick tissues, requiring excessive milling time and discarding most of the sample. To overcome these limitations, cryo-lift-out has been developed—a technique in which a micromanipulator physically extracts a chunk of interest from deep within the tissue and transfers it to a dedicated grid for final thinning. This approach bypasses the thickness barrier and enables site-specific analysis of internal structures. This review systematically traces the evolution of cryo-lift-out from its origins in materials science to its adaptation for biological tissues. In room-temperature lift-out, reliable attachment is achieved by gas-injection system (GIS)-assisted metal deposition. Transferring this approach to cryogenic conditions proved challenging because precursor gases condense on all cold surfaces, leading to contamination and poor adhesion. The development of copper-assisted redeposition marked a critical turning point: instead of relying on gas deposition, this method uses ion-beam sputtering to deposit copper atoms at the needle-chunk interface, creating a strong, low-contamination bond. This innovation has enabled robust cryo-lift-out workflows and paved the way for serial lift-out, in which multiple consecutive lamellae are prepared from a single tissue chunk, substantially increasing throughput and enabling volumetric imaging. Despite these advances, several technical challenges remain. Curtaining effects caused by uneven chunk surfaces can introduce artifacts into tomograms, requiring careful optimization of milling parameters and protective coating. The cryo-adhesion step still demands precise control of beam angle, needle positioning, and milling depth, making the process highly operator-dependent. Additionally, the choice of grid geometry is critical. Custom-designed grids with double-sided attachment improves stability and offer better compatibility with cryo-ET tilt series. Automation, which has greatly improved room-temperature lift-out, has not yet been achieved for cryo-lift-out due to the complexity of handling heterogeneous biological tissues and the need for real-time adaptation. Future progress will likely focus on integrating cryo-lift-out with volume electron microscopy to correlate ultrastructure across scales, developing intelligent control systems to reduce user intervention, and extending the technology to challenging samples such as plant tissues and some material science samples for interface study. A systematic analysis of the cryo-lift-out technique clarifies the key limiting factors for its large-scale application and lays a foundation for methodological refinement and technological innovation. By consolidating recent advances and identifying remaining bottlenecks, this review aims to support the broader adoption of cryo-lift-out and accelerate the development of tissue-scale in situ structural biology.

The TMEM41B scramblase and its regulatory partner CLCC1 initiate lipid flux by equilibrating newly synthesized phospholipids across the endoplasmic reticulum (ER) bilayer, a fundamental process required for diverse events ranging from membrane biogenesis to bulk lipid supply. Loss of CLCC1/TMEM41B causes ER bilayer imbalance, which induces giant ER-enclosed lipid droplets (geLDs) and drives rapid progression into severe metabolic-dysfunction-associated steatohepatitis (MASH). Combining both human cell lines and mouse models, we herein reveal CLCC1 to be the long-missing client of the luminal torsin ATPases, which selectively engage oligomerized CLCC1 at sites of ER bilayer imbalance. Mice hepatic torsinA inactivation triggers geLD formation amid disrupted lipoprotein biogenesis and severe MASH, closely phenocopying CLCC1/TMEM41B deficiency. Mechanistically, torsins act as assembly-promoting ATPases that drive CLCC1 oligomerization for its recruitment to imbalanced bilayers. Remarkably, ectopic CLCC1 expression reverses cellular and systemic lipid disorders arising from hepatic torsinA deficiency. Hence, torsin ATPases emerge as fundamental regulators that organize CLCC1 and the downstream TMEM41B scramblase to govern lipid partitioning and membrane homeostasis.

With the advancement of the Materials Genome Initiative, high-throughput computation has become central to accelerating materials discovery. However, conventional first-principles workflows are cumbersome and error-prone. Existing high-throughput tools, while efficient at batch job submission, lack intelligence: they cannot automatically plan tasks based on scientific objectives or dynamically adapt workflows according to intermediate results. To address these limitations, this paper proposes and implements HTC-Claw, an intelligent high-throughput computational platform built upon the OpenClaw framework. The key innovations of HTC-Claw are: 1) An agent-based framework for automatic decomposition of high-level research goals into parallelizable task sets; 2) A closed-loop execution engine that integrates real-time analysis and reporting; 3) Adaptive decision-making and workflow iteration capabilities based on intermediate results; and 4) A decoupled, modular architecture that separates the scheduling system from functional modules, enhancing extensibility and robustness. Case studies demonstrate that HTC-Claw enables an intelligent, end-to-end workflow from user intent to final reporting in materials exploration

Long-term natural evolution has selected glycolysis as the major metabolic mode to generate energy and cellular building blocks for rapid cell growth in eukaryotes such as yeast; however, glycolysis lacks a sufficient NADPH supply to drive the biosynthesis of reduced chemicals. Industrially feasible cell factories often require the most energy-economical pathway for the high-level production of target products. In this study, we successfully established a pentose phosphate pathway-dominant, glycolysis-minimized central metabolic mode in yeast, which significantly improved the cellular energy status compared with glycolysis and provided a balanced supply of ATP and NADPH. More importantly, we discovered a global carbon metabolism regulator, which drives metabolic flux toward glycolysis for energy generation, and whose disruption relieved the tight regulation of metabolic flux distribution and significantly enhanced the cellular energy status, which in turn improved the production of the energy-intensive molecules free fatty acids (FFAs) by 63%. The final engineered strain produced FFAs at a titer of 41.7 g/l, the highest titer reported for yeast fermentation. Our work provides valuable insights into the metabolic regulatory mechanisms of microorganisms.

The SPFH (stomatin, prohibitin, flotillin, and HflK/C) family proteins are proposed scaffolds for organizing functional membrane microdomains (FMMs) on various cellular membranes. Erlin1 and Erlin2, two endoplasmic reticulum (ER)-residing SPFH members, as heteromeric complexes, participate in ER-associated protein degradation (ERAD). However, the mechanisms underlying Erlin-mediated FMM organization and ERAD regulation remain poorly understood. Here, through cryoelectron microscopy (cryo-EM), we find that the human Erlin1/2 complex forms a 26-mer cage assembly, defining a nanometer-sized microdomain on the luminal leaflet. The intramembrane region of each subunit constitutes a specific phosphatidylinositol-binding pocket. ER proteins can be recruited to both the interior and exterior of these cages. By caging cargoes, the Erlin1/2 complex physically secludes them from their substrates or binding partners, conferring another layer of regulation on their functions. Moreover, individual cages can cluster to organize FMMs of different sizes. These dynamic properties underscore a general regulatory role of Erlin1/2 in various ER-related biological processes, including coronaviral replication.

Mitochondrial function critically depends on protein quality control systems, with the m-AAA protease playing a key role at the inner mitochondrial membrane (IMM). The evolutionarily conserved prohibitins (PHBs) are essential modulators of this protease across species, yet the molecular mechanisms remain unclear. Here, we present the cryo-EM structure of the Chaetomium thermophilum PHB (CtPHB) complex, revealing a cage-like assembly composed of 11 copies of PHB1/PHB2 heterodimers. Electron microscopic and biochemical analyses suggest that m-AAA proteases are enclosed within the PHB complex through interactions mediated by their SPFH-interacting motif (SIM) exposed in the intermembrane space. Further in situ cryo-ET directly visualizes these cage-protease assemblies in native mitochondria. Disruption of their interface leads to elevated m-AAA protease activity and diminished mitochondrial stress resistance. These data establish PHB complexes as spatial organizers that compartmentalize m-AAA proteases in membrane microdomains to fine-tune proteolytic homeostasis. Our findings reveal the critical role of the PHB complex in maintaining mitochondrial proteostasis, providing a unified mechanistic model to explain and reconcile the pleiotropic and often contradictory phenotypes of PHB and m-AAA protease in mitochondrial physiology and various disease conditions.

Ribosome biogenesis and mRNA translation are fundamental cellular processes regulated by a diverse set of protein factors, including GTPases. In bacteria, while several GTPases are known to participate in ribosome assembly, their precise mechanisms in subunit maturation and their potential roles in translation regulation remain largely elusive. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of native pre-50S assembly intermediates and 70S translating ribosomes, isolated via epitope-tagged GTPases from engineered Escherichia coli cells at resolutions of 2.3-4.4 Å. These structures elucidate how three GTPases, YihA, EngA and ObgE, act as successive placeholders to mediate rRNA folding and to coordinate the correct timing of the maturation of different functional blocks within the large ribosomal subunit. Furthermore, our data identify several previously unrecognized 70S translational complexes bound by the GTPases EngA and BipA—factors traditionally regarded as assembly factors, thereby uncovering their regulatory role in bridging ribosome assembly and translation initiation. Collectively, our findings delineate a GTPase-mediated surveillance system that continuously monitors the assembly of ribosomal subunits and translation adversity, thereby safeguarding protein synthesis and maintaining proteome homeostasis.

Flotillin-1 and flotillin-2 form hetero-oligomers to create flotillin membrane microdomains essential for endocytosis and protein sorting. However, the mechanisms of flotillin oligomerization and microdomain organization remain incompletely understood. Here, we present the cryo-EM structure of human flotillin complex, showing that flotillin-1 and -2 form a 44-mer, membrane attached, and dome-shaped structure that defines a 30-nm circular membrane domain. The cryo-ET data demonstrates that while attached to the cytoplasmic leaflet, flotillin complexes possess intrinsic structural plasticity in situ on the native membrane. Each flotillin complex may represent a fundamental unit of membrane microdomains, with their clustering enabling the formation of larger and more elaborate domains. We further reveal that phosphorylation at residues Y160 (flotillin-1) and Y163 (flotillin-2) may act as a molecular switch to modulate complex assembly, potentially regulating its function in endocytosis. These findings demonstrate the molecular mechanism of flotillin-mediated membrane segregation and microdomain formation, and suggest a previously unrecognized role of flotillin in sequestrating membrane proteins.

Flexible ionic thermoelectric (i-TE) quasi-solid cells provide an electrolyte leak free, easy-operation and mechanically adaptable route to low-grade heat harvest. However, limited by freezing and evaporation, the hydrogel-based thermocell exhibits environmental instability and has difficulty functioning at low temperatures. Herein, we develop a high-performance cryo-temperature i-TE eutectogel using a water-containing deep eutectic solvent (DES) strategy. The water containing DES is intentionally utilized to modulate the solvent shell structure of redox couple and expand the operation temperature range of i-TE eutectogel cell. Moreover, appropriate amount of water can increase the solubility of [Fe(CN)6]3-/4- , while maintaining the eutectic properties of DES. Subsequently, a double-network polymer gel is constructed to prevent liquid electrolyte leakage and provide the flexibility and operability of the i-TE cell. After synergistic optimization of the solvent and the polymer network, the Seebeck coefficient of the fabricated i-TE eutectogel cell reaches 2.06 mV/K. In a 20-cell prototype module, the current and voltage at Delta T = 8.3 K are 0.033 mA and 0.33 V, respectively. The DES endows this cell wide operable temperature ranging from-60 degrees C to 80 degrees C. The anti-freezing capability of this eutectogel cell offer a new strategy for fabricating gel-based thermocell required in frigid regions.

Random lasers (RLs) with a simple structure and low-cost properties have been recognized as an ideal analytical platform and are still challenging for liquid detecting, remaining beset for low sensitivity, complicated operation, and large analyte consumption. Here, inspired by a microfluidic sensor, a microtubule structured random laser for multifunctional sensing is demonstrated. The random laser is achieved resorting to a curly PMMA film with gain and scatterers embedded in it. By coupling the high-order WGM with a weak random scattering mode, a coherent random laser with a low threshold of 0.62 MW cm-2 and a high Q factor of 4700 is obtained. The sensing process has been demonstrated based on two kinds of representative analytes of sucrose solution and TiO2 NP suspension, which respond to the variation of gain and scattering. The RL sensor features fast detection, easy operation, and low cost, which may provide a new approach for their further applications in analytical microfluidic chip and disposable/pocket analytical instruments.

Ribosome biogenesis is a complex and error-prone process, necessitating quality control mechanisms to degrade defective pre-ribosomal intermediates. In this issue of Molecular Cell, Akers et al.1 report the identification of a previously uncharacterized quality control pathway named ribosome assembly surveillance pathway (RASP), which functions to eliminate aberrant "dead-end" pre-60S assembly intermediates.

Utilizing plasmonic hot-electrons to drive chemical reactions is an emerging technique for converting light energy to chemical energy. Developing effective strategies to implement these processes under normal sunlight is a crucial step toward practical applications but remains challenging. Herein, ordered poly(ionic liquid) (PIL) nanocone arrays with optimized geometry were fabricated, which function as an omnidirectional light-harvesting antenna platform to facilitate hot-electron-mediated chemistry. It was found that due to the unique ion-exchange properties involved in PIL, it enables the facile incorporation of various plasmonic metal nanoparticles (Au, Ag, or Cu) into PIL nanocones through an anion-exchange process followed by reduction, showing great flexibility and extendibility. Moreover, hot carriers with different energies can be efficiently generated owing to the enhanced light absorption of nanoparticles (NPs) embedded within nanocone arrays across a wide spectrum. More importantly, various metal cations can be further reduced by the generated hot-electrons of embedded plasmonic NPs, forming a series of bimetallic patchy NPs under normal sunlight. The power of our strategy was exemplified by the formation of "catalytic patch-plasmonic core" structures combined with a light-harvesting PIL array matrix. This integrated catalytic system exhibits enhanced photocatalytic performance using only natural outdoor sunlight, highlighting the critical role of PIL nanocone arrays as light-harvesting antennas in enhancing hot-carrier generation efficiency. We postulate that PIL nanocone arrays would hold great potential for developing plasmon-based photocatalytic systems.

The Sec translocon is vital for guiding membrane protein insertion into lipid bilayers. The insertion and folding processes of membrane proteins are poorly understood. Here, we report cryo-electron microscopy structures of multi-spanning membrane proteins inserting through the SecY channel, the Sec translocon in prokaryotes. The high-resolution structures illustrate how bulky amino acids pass the narrow channel restriction. Comparison of different translocation states reveals that the cytoplasmic and extracellular cavities of the channel create distinct environments for promoting the unfolding and folding of transmembrane segments (TMs), respectively. Released substrate TMs are either flexible or stabilized by an unexpected hydrophilic groove between TM3 and TM4 of SecY. Disruption of the groove causes global defects in the folding of the membrane proteome. These findings demonstrate that beyond its role as a passive protein-conducting channel, the SecY translocon actively serves as a chaperone, employing multiple mechanisms to promote membrane protein insertion and folding.

Self-assembly is a fundamental property of living matter that drives the three-dimensional organization of cell collectives such as tissues and organs. Here, the co-assembly of synthetic and natural cells is leveraged to create hybrid living 3D cancer cultures. We screen a range of synthetic cell models for their ability to form augmented tumoroids with artificial but controllable micro-environments, and show that the balance of inter- and extracellular adhesion and synthetic cell surface tension are key material properties driving integrated co-assembly. We demonstrate that synthetic cells based on droplet-supported lipid bilayers can establish artificial tumor immune microenvironments (ART-TIMEs), mimicking immunogenic signals within tumoroids and eliminating the need to integrate complex living immune cells. Using the ART-TIME approach, we identify a AhR-ARNT-mediated co-signaling mechanism between PD-1 and CD2 as a driver in immune evasion of pancreatic ductal adenocarcinoma. Our study advances the field of hybrid organoid engineering, offers opportunities for the construction and modelling of artificial tumour environments, and marks a step towards the design of functional living/non-living cytomimetic materials.

Phycobilisomes (PBSs) are the major light-harvesting complexes in the cyanobacteria and red algae and they consist of a central core and peripheral rods that are attached to the core. The PBS cores contain 2–5 allophycocyanin cylinders that are organized by ApcE. At the present, structures of PBS with tricylindrical and pentacylindrical cores have been determined while the structure of the PBS with a bicylindrical core is yet to be revealed. Here we report the cryo-EM structure of PBS with bicylindrical core from Synechococcus elongatus PCC 7942 (Synechococcus 7942) at an overall resolution of approximately 3 Å. Similar to the PBS with a tricylindrical core, six peripheral rods are attached to the core by the rod-core linker protein CpcG in the PBS of Synechococcus 7942 even though the core lacks the top AP cylinder, which is important for the attachment of peripheral rods to the tricylindrical cores. We found that the C-terminus of ApcE in the Synechococcus 7942 was involved in interacting with both CpcG and CpcB of a top peripheral rod, compensating for the absence of the top AP cylinder of the core and maintaining PBS stability. Analysis of the bilin distribution reveals that distance of excitation energy transfer from top peripheral rods to the terminal emitters is approximately 15

Eukaryotic ribosome biogenesis is an energy-consuming process involving many ATPase-driven steps. In yeast, AAA+ protein Drg1 releases an assembly factor Rlp24, a placeholder for Rpl24, from pre-60S particles just exported to cytosol. The equivalent process in human cells involves SPATA5 (Drg1 homolog) and additional factors. However, the mechanistic details remain unclear. Here we reveal that SPATA5 forms a 4:2:2:2 complex with SPATA5L1, C1orf109, and CINP. This complex features an N-terminal ring made of C1orf109, CINP and NTDs of SPATA5/SPATA5L1, and two hexameric AAA+ ATPase rings. Intriguingly, a conserved cysteine C672 in the P-loop of SPATA5 is sulfinylated, generating an inactive conformation incompatible with ATP binding. We also obtained a cryo-EM structure of pre-60S-bound SPATA5 complex. Different from yeast, the recognition of the pre-60S particle is mediated by human-specific factor CINP, through two distinct sets of interactions: one with GTPBP4 and the other with ES27A. Taken together, these data provide structural basis for understanding the cytoplasmic maturation of the pre-60S, and reveal human-specific features that might be harnessed for therapeutic purposes.

The SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) family of proteins are key scaffolding components involved in the organization of functional membrane microdomains (FMMs) across various subcellular membranes, including those of the endoplasmic reticulum (ER) and mitochondria, which are characterized by extremely low saturated lipid content. Among this protein family, the erlin1/2 complex is specifically located on the ER membrane. Previous studies have shown that the erlin1/2 complex plays essential roles in the ER-associated degradation (ERAD) pathway, mediating the ubiquitin-dependent degradation of various proteins such as inositol 1,4,5-trisphosphate receptors (IP3Rs), important calcium ion transporters on the ER membrane. In this study, we determined the single-particle cryo-electron microscopy (cryo-EM) structure of the erlin1/2 complex under different detergent conditions. Our findings reveal that the erlin1/2 complex forms a 26-mer cage-like structure, composed of alternating erlin1 and erlin2 subunits. The erlin1/2 complex could recruit various types of proteins on both the interior and exterior surfaces of the cage. By caging cargo proteins, the erlin1/2 complex physically seclude them from their binding partners, leading to a potential halt of their function. Moreover, individual cages can further interact with one another, facilitating the organization of FMMs of different sizes on the ER membrane. These dynamic properties may play a general and critical role in various processes occurring on the ER, including viral replication, positioning the erlin1/2 complex as a promising new target for antiviral drug development. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 92354306

In eukaryotes, the origin recognition complex (ORC) promotes the assembly of minichromosome maintenance 2 to 7 complexes into a head-to-head double hexamer at origin DNA in a process known as replication licensing. In this study, we present a series of cryoelectron microscopy structures of yeast ORC mutants in complex with origin DNA. We show that Orc6, the smallest subunit of ORC, utilizes its transcription factor II B-B domain to orchestrate the sequential binding of ORC to origin DNA. In addition, Orc6 plays the role of a scaffold by stabilizing the basic patch (BP) of Orc5 for ORC to capture and bend origin DNA. Importantly, disrupting DNA bending through mutating three key residues in Orc5-BP impairs ORC's ability to promote replication initiation at two points during the pre-RC assembly process. This study dissects the multifaceted role of Orc6 in orchestrating ORC's activities on DNA and underscores the vital role of DNA bending by ORC in replication licensing.