Methanogens are central to global carbon cycling and among the largest biological sources of methane, a potent greenhouse gas1. At the heart of their energy metabolism lies the Hdr-Vhu-Fwd super-assembly, which couples H2 oxidation with CO2 reduction through flavin-based electron bifurcation. Here we present the cryogenic electron microscopy structure of the Hdr-Vhu-Fwd super-assembly from Methanococcus maripaludis, revealing an 8 MDa complex comprising 252 polypeptide chains and over 600 redox cofactors. Cryo-electron tomography further support that this super-assembly forms an intact structure within the cytoplasm of intact cells. This architecture comprises two hexameric HdrABC-Vhu rings linked by a tetrameric FwdF core, forming a continuous, circular electron chain. In this unique arrangement, 12 polyferredoxin subunits (VhuB) connect the Vhu-Hdr and Fwd complexes, thereby coupling electron bifurcation with CO2 reduction and directly linking the last and the first step of methanogenesis. Moreover, we identify a modular variant of the complex in which the [NiFe]-hydrogenase Vhu is substituted by tungsten-containing formate dehydrogenase (FdhAB), indicating flexible integration of electron-input modules facilitating metabolic adaptation under diverse environmental conditions2. Analysis of the taxonomic distribution reveals that this architecture is specific to class I methanogens and is distinct from the smaller Hdr-Fmd complex of class II3. Together, our study reveals that the the Hdr-Vhu-Fwd super-assembly has a modular and adaptable bioenergetic assembly, suggesting a lineage-specific architecture to adapt to diverse anaerobic niches.

Cyanobacteria are major contributors to global photosynthesis and are intensively studied for sustainable green H 2 production. Central to this process is the bidirectional [NiFe]-hydrogenase HoxEFUYH, yet its physiological redox partners have remained unresolved. Ferredoxin, NAD(H), and NADP(H) have been proposed as partners, but the lack of active enzyme preparations has prevented a definitive assignment. Here, we purified the intact HoxEFUYH complex from Synechocystis sp. PCC 6803 under strictly anaerobic conditions and reveal its function as both a bifurcating and confurcating hydrogenase. During H 2 uptake, HoxEFUYH utilizes NAD + and oxidized ferredoxin, whereas H 2 production strictly requires both NADH and reduced ferredoxin; NADPH does not support either reaction. Combining high-resolution cryo-electron microscopy with biochemical and spectroscopic analyses, our data reveal that an flavin-containing reductase module is electronically connected to the catalytic [NiFe]-hydrogenase core through an extended chain of iron-sulfur clusters, defining the structural basis for bifurcating and confurcating electron flow. These findings fundamentally revise the physiological role of HoxEFUYH by showing that photosynthetic H 2 production does not rely solely on photosynthetic electrons but instead couples reduced ferredoxin from the light reaction with NADH derived from “dark” carbohydrate oxidation. This requires reassessment of current strategies for green H 2 production in cyanobacteria.

Abstract The fixation of dissolved inorganic carbon (DIC) such as CO2 and bicarbonate is fundamental to the global primary production. Many autotrophs depend on a diversity of CO2-concentrating mechanisms (CCMs) to overcome the inefficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the limited supply of DIC. While cyanobacterial CCMs are well characterized, analogous systems in chemolithoautotrophs, specifically active DIC uptake systems have long been overlooked. Here, we present the cryo-EM analysis of DAB2, an essential membrane protein complex for CO₂ uptake in Halothiobacillus neapolitanus. The cytoplasmic subunit DabA2 displays a β-carbonic anhydrase-like fold, while the transmembrane subunit DabB2 resembles the proton-conducting subunits of respiratory Complex I. Purified DAB2 binds CO₂ independent of protonmotive force (PMF); however, did not spontaneously hydrate CO2. Structural analysis reveals a deeply buried active site only accessible via gated substrate tunnels, suggesting substrate access and catalysis are tightly regulated. A distinct transmembrane helix of DabA2 forms the proton pathway and potentially couples proton translocation to catalysis. These features define a vectorial CO2 hydration mechanism that prohibits reverse bicarbonate dehydration. Our findings establish DAB2 as a prototype of a family of PMF-driven carbonic anhydrases, elucidating a distinct strategy for CO₂ capture in non-photosynthetic autotrophs.

The Rnf complex is the primary respiratory enzyme of several anaerobic prokaryotes that transfers electrons from ferredoxin to NAD+ and pumps ions (Na+ or H+) across a membrane, powering ATP synthesis. Rnf is widespread in primordial organisms and the evolutionary predecessor of the Na+-pumping NADH-quinone oxidoreductase (Nqr). By running in reverse, Rnf uses the electrochemical ion gradient to drive ferredoxin reduction with NADH, providing low potential electrons for nitrogenases and CO2 reductases. Yet, the molecular principles that couple the long-range electron transfer to Na+ translocation remain elusive. Here, we resolve key functional states along the electron transfer pathway in the Na+-pumping Rnf complex from Acetobacterium woodii using redox-controlled cryo-electron microscopy that, in combination with biochemical functional assays and atomistic molecular simulations, provide key insight into the redox-driven Na+ pumping mechanism. We show that the reduction of the unique membrane-embedded [2Fe2S] cluster electrostatically attracts Na+, and in turn, triggers an inward/outward transition with alternating membrane access driving the Na+ pump and the reduction of NAD+. Our study unveils an ancient mechanism for redox-driven ion pumping, and provides key understanding of the fundamental principles governing energy conversion in biological systems.

Methanogenic archaea emit ~1 Gt of methane annually, impacting global carbon cycling and climate. Central to their energy metabolism is a membrane-bound, sodium-translocating methyltransferase complex: the N⁵-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr). It couples methyl transfer between two methanogen-specific cofactors with sodium ion transport across the membrane, forming the only energy-conserving step in hydrogenotrophic methanogenesis. Here, we present a 2.1 Å single-particle cryo-EM structure of the Mtr complex from Methanosarcina mazei. The structure reveals the organization of all catalytic subunits, embedded archaeal lipids and the sodium-binding site. Most strikingly, we discover MtrI, a previously unannotated small open-reading frame encoded protein ( < 100 aa) found within the order of Methanosarcinales that binds both the top of the sodium-channel and cytosolic domain of MtrA via its cobamide cofactor in response to oxygen exposure. This interaction likely prevents sodium leakage and stabilizes the complex under oxidative conditions, revealing an unexpected regulatory mechanism in methanogen energy conservation. Here, the authors present the cryoEM structure of the sodium-translocating methyltransferase (Mtr) complex from Methanosarcina mazei. Along with providing catalytic insights, they identify MtrI, an unannotated small protein, bound to the Mtr complex in a redox-dependent manner.

Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei , it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK 1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA 1 ) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA 1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA 1 . The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK 1 . The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK 1 and crucially depends on the presence of 2-OG.

ABSTRACTWhen faced with a DNA double strand break, cells activate an elaborate signaling cascade called the DNA damage response to protect genomic integrity. To identify novel factors that modulate the DNA damage response to DNA double strand breaks, we performed an epistatic miniarray profile (E-MAP) analysis of Mec1 and Rad53, two essential kinases that coordinate the DNA damage response in budding yeast. Through this analysis, we discovered a genetic interaction between the kinase module (CKM) of the Mediator of transcription and Rad53. We find that all four subunits of the CKM, as well as CKM’s kinase activity are critical for cell cycle re-entry following a DNA break, whereas the core Mediator subunits are dispensable. Notably, CKM mutants do not impair DNA repair by homologous recombination or confer sensitivity to DNA damaging reagents, suggesting that CKM specifically impinges on DNA damage signaling. In support of this, we find that Rad53 and CKM physically interact in response to DNA damage. Following the induction of a DNA break, CKM is a critical regulator of global transcription inhibition. In addition to this global effect, we illustrate that CKM functions locally at DNA breaks together with the core Mediator. In the absence of catalytically active CKM, the CKM-Mediator complexes at DNA breaks are replaced by RNAPII. Taken together, our results reveal a previously uncharacterized role for CKM in the DNA damage response.

Methyl-coenzyme M reductase (MCR) is the enzyme responsible for nearly all biologically generated methane 1 . Its active site comprises coenzyme F 430 , a porphyrin-based cofactor with a central nickel ion that is active exclusively in the Ni(I) state 2,3 . How methanogenic archaea perform the reductive activation of F 430 represents a major gap in our understanding of one of the most ancient bioenergetic systems in nature. Here we purified and characterized the MCR activation complex from Methanococcus maripaludis . McrC, a small subunit encoded in the mcr operon, co-purifies with the methanogenic marker proteins Mmp7, Mmp17, Mmp3 and the A2 component. We demonstrated that this complex can activate MCR in vitro in a strictly ATP-dependent manner, enabling the formation of methane. In addition, we determined the cryo-electron microscopy structure of the MCR activation complex exhibiting different functional states with local resolutions reaching 1.8–2.1 Å. Our data revealed three complex iron–sulfur clusters that formed an electron transfer pathway towards F 430 . Topology and electron paramagnetic resonance spectroscopy analyses indicate that these clusters are similar to the [8Fe-9S-C] cluster, a maturation intermediate of the catalytic cofactor in nitrogenase. Altogether, our findings offer insights into the activation mechanism of MCR and prospects on the early evolution of nitrogenase.

Photosystem II (PSII) assembly is a stepwise process in which intermediate complexes with auxiliary proteins are transiently formed to allow efficient de novo biogenesis or repair of damaged PSII. In par-ticular, the role of extrinsic PSII subunits (PsbO, PsbU, PsbV) and auxiliary proteins such as Psb27 for the formation and photoactivation of the Mn4O5Ca cluster, which catalyzes the unique water splitting reaction in mature PSII, remains unclear. Using cryo-electron microscopy, we have determined the structure of two novel late-stage PSII assembly intermediates. In contrast to previous studies, the result-ing monomeric PSII complexes contain both PsbJ and Psb27 and exhibit a fully mature acceptor side, while the oxygen evolving complex (OEC) is still in an immature state. The second complex additionally associates with the late-acting assembly factor Psb32 and the extrinsic subunit PsbV. While Psb32 has received little attention, its proposed role in the complex challenges the previous assumption that all extrinsic subunits associate spontaneously, as well as the notion that PsbO initiates binding and solely drives OEC formation. Our structures of the Psb27-PSII and Psb32-PSII intermediates provide novel insights, how structural changes of C-termini of the D1 and D2 core proteins regulate maturation of the OEC and how the catalytic side is prepared for binding of the Mn4O5Ca cluster. The Psb32-PSII complex potentially represents the final PSII assembly intermediate that precede the incorporation and photoactivation of the Mn4O5Ca cluster, allowing us to explain the final steps in the PSII biogenesis and assembly pipeline in great detail, as only the two extrinsic subunits PsbO and PsbU are missing. ### Competing Interest Statement The authors have declared no competing interest. * AC : Affinity chromatography Chl a : Chlorophyll a Cyt : Cytochrome LMWP : Low molecular weight protein MALDI-ToF : Matrix assisted laser desorption/ionization-time of flight MS : Mass spectrometry MDFF : molecular dynamics flexible fitting OEC : Oxygen evolving complex PQ : Plastoquinone PSII : Photosystem II RC : Reaction center RCC : Reaction center complex ROS : Reactive oxygen species TST : Twin-Strep tag Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64

Methanogenic archaea contribute 1–2 Gt of methane annually, impacting both global carbon cycling and climate. Central to their energy metabolism is a membrane-bound, sodium-translocating methyltransferase complex: the N⁵-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr complex), which catalyzes the methyl transfer between two methanogen specific cofactors. This exergonic methyl transfer step is coupled with a vectorial sodium ion transport from the cytoplasm to the cell exterior and is the only energy conserving step in hydrogenotrophic methanogenesis. Here, we present a 2.1 Å single-particle cryo-EM structure of the full Mtr complex from Methanosarcina mazei . Our structural model encompasses the entire complex, reveals the arrangement of archaeal phospholipids, the architecture of the sodium ion binding site, and the structure and interactions of all catalytic subunits. Most strikingly, we discover and characterize MtrI, a previously unannotated small open reading frame (small ORF), encoded protein (<100 aa) conserved across the order of Methanosarcinales. MtrI binds to the cytoplasmic domain of MtrA in response to oxygen exposure, suggesting a role in oxygen stress response and protection. By binding on top of the sodium channel and anchoring to the cobamide cofactor in MtrA’s cytoplasmic domain, MtrI might prevent sodium leakage and inhibit MtrA-CoM turnover. These findings offer new insights into methanogen energy conservation and uncover a potential adaptive response to oxygen exposure, expanding our understanding of methanogen survival strategies under oxidative stress. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, 101075992 Deutsche Forschungsgemeinschaft, SCHU 3364/1-1 Deutsche Forschungsgemeinschaft, RTG 2937 Deutsche Forschungsgemeinschaft, SCHM1052/20-2

The proteasome activator PA200 binds to the catalytic core of the proteasome, the 20S, and activates its proteolytic activities. The cellular function of PA200 is poorly understood and appears to be cell type and differentiation specific. Recent evidence suggests that PA200 not only binds to the standard 20S (s20S) proteasome but also to the specialized immunoproteasome (i20S) which plays a key role in anti-viral and anti-tumor immunity. We here investigated the interaction of PA200 and the immunoproteasome in detail. We show the very first cryo-EM structures of the singly- and doubly-capped i20S-PA200 complexes that revealed no major difference regarding the first binding event of PA200 to the i20S vs. the s20S. However, first PA200 binding triggered a subtle and long range allosteric bending of the i20S barrel which was not seen in the s20S-PA200 complexes. This resulted in major structural rearrangements in the opposite unbound α ring - the displacement of atoms up to 5.4 Å and the increase in its outer diameter - thereby increasing the occupancy of the second PA200 binding site. Mass photometry confirmed higher occupancy of PA200 to the i20S versus the s20S. Binding of PA200 to the i20S enhanced proteasomal activation compared to the s20S. Co-expression of PA200 and the i20S in cells and tissues, however, is restricted but their interaction is favored upon co-expression. The expression of PA200 and the catalytic subunits of the i20S is differentially regulated depending on the cellular context. Our data also suggest that PA200 has the potential to regulate i20S gene expression whereas the i20S has no effects on PA200 expression. Overall, this work sheds new light on the interaction of PA200 with the i20S from a structural, mechanistic and cellular point of view. Importantly, we identify PA200 as a key regulator of the i20S whenever PA200 and the catalytic subunits of the i20S are co-expressed in the same cell. ### Competing Interest Statement The authors have declared no competing interest.

TGFβ-signaling regulates cancer progression by controlling cell division, migration, and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision-making toward specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing, and morphological assays to quantitatively link signaling, gene expression, and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that most of the TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision-making.

Marburg virus (MARV) causes lethal hemorrhagic fever in humans, posing a threat to global health. We determined by cryogenic electron microscopy (cryo-EM) the MARV helical ribonucleoprotein (RNP) complex structure in single-layered conformation, which differs from the previously reported structure of a double-layered helix. Our findings illuminate novel RNP interactions and expand knowledge on MARV genome packaging and nucleocapsid assembly, both processes representing attractive targets for the development of antiviral therapeutics against MARV disease. Zinzula et al. reconstituted the helical ribonucleoprotein complex of Marburg virus in vitro, and determined its structure by cryo-electron microscopy in a single-layer conformation that recapitulates the assembly of authentic filovirus particles.

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assembly states and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

This study developed and evaluated a prototype for augmented reality (AR) interaction among multiple users sharing a three-dimensional hologram within a shared physical space. A systems requirements analysis was conducted to determine necessary interactions, followed by an iterative implementation plan with testing at each step. The application for the HoloLens 2 (HL2) headset was developed using Unity for 3D design, C# for programming, and Photon for multiplayer capabilities. The prototype was evaluated in a user study utilizing custom and NASA TLX questionnaires, comparing participants' self-reported task success with recorded data to assess the accuracy of selfassessments. The prototype effectively facilitated multi-user interactions within a shared environment, achieving acceptable synchronization for two users. Most participants successfully completed tasks, with TLX scores indicating low cognitive and mechanical load during AR task execution. This study demonstrates AR's viability for displaying and manipulating complex three-dimensional structures within a collaborative medical context among multiple individuals in the same physical space.