Biology subjects

Namba, K.

Publications and source records attributed to Namba, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Thermal fractuations assist high-speed rotation of the bacterial flagellar motor at low load

The bacterial flagellar motor is a proton-driven rotary nanomachine that converts ion flow into mechanical motion. Proton-coupled rotation of the MotA5-MotB2 stator complex generates torque, yet how this process drives high-speed rotation remains unclear. Here we use single-molecule nanophotometry to resolve stepwise rotation under low-load conditions. Lowering intracellular pH selectively prolongs dwell times without affecting step durations, indicating that proton dissociation triggers torque generation. Structural analysis further suggests that the rotor disengages from the stator before completing the elementary step angle (~11 degree), implying that the power stroke alone is insufficient. Notably, the rotational diffusion coefficient of stator-less motors closely matches that inferred from wild-type motors at low load. These findings support a model in which intrinsic thermal fluctuations compensate for the limited reach of the power stroke, enabling rapid rotation. Our results reveal a hybrid mechanism in which ion-driven conformational changes bias stochastic motion to achieve efficient energy transduction.

biophysics↗

bioRxiv2026

Structural characterization of a pseudaminic acid-modified lateral flagellar filament from Vibrio alginolyticus

Bacterial flagellar filaments are often modified by glycans, but the structural basis and physiological significance of flagellin glycosylation remain poorly understood in many species. Vibrio alginolyticus produces lateral flagella for surface-associated motility in viscous environments, with LafA forming its filament as flagellin. A maf homolog encoding a putative flagellin glycosylation factor is located immediately downstream of lafA, suggesting that the lateral filament is glycosylated. To investigate this possibility, we purified the lateral flagellar filament from V. alginolyticus and determined the structure at 2.37 [A] resolution by electron cryomicroscopy. Upon model building of LafA in the map, we identified additional densities connected to five serine residues, possibly corresponding to O-linked pseudaminic acid modifications. Mass spectrometric analyses identified these modifications as pseudaminic acid attached to Ser148, Ser173, Ser183, Ser189, and Ser197. Deletion of maf abolished lateral flagella formation and motility, and these defects were restored by complementation with maf. These results demonstrate that the Vibrio lateral flagellar filament is extensively modified by pseudaminic acid and that Maf is required for filament formation. Our findings provide the first structural insight into the glycosylation of Vibrio lateral flagellar filament and establish a framework for understanding the role of flagellin glycosylation in surface-associated motility.

microbiology↗

bioRxiv2026

Structural insights into fosfomycin efflux by a streptococcal ABC transporter

Gram-positive bacteria encode a broad array of ABC transporters that mediate substrate translocation across the cell membrane, with some contributing to their survival under environmental stresses such as antimicrobial exposure. While several of these transporters have been shown to exhibit multidrug efflux activity, the functional roles of many others remain unknown. Here, using an efflux pump screen in the opportunistic human pathogen Streptococcus pneumoniae, we identified a previously uncharacterized type IV ABC transporter (FoeAB) that confers resistance to the antibiotic fosfomycin. We show that purified FoeAB mediates fosfomycin transport in a liposome-reconstituted system and provide evidence that it functions as a multidrug efflux pump with substrate preferences distinct from those of known efflux pumps. Furthermore, we present cryogenic electron microscopy (cryo-EM) structures of FoeAB in inward- and outward-facing states, which reveal conformational changes associated with nucleotide binding and identify residues important for substrate transport. Collectively, these findings expand the known repertoire of antibiotic-exporting ABC transporters in Gram-positive bacteria and provide structural insight into its transport mechanism. SIGNIFICANCEEfflux pumps often play major roles in antibiotic resistance in clinically important pathogens, yet many of these transporters remain poorly defined. In this study, we identify and mechanistically characterize FoeAB, a previously unrecognized type IV ABC transporter in streptococcal species that mediates efflux of the antibiotic fosfomycin. By combining functional assays with structural studies of FoeAB in multiple conformational states, we propose a model for how this transporter recognizes its substrate and harnesses nucleotide-driven conformational changes to mediate substrate export. Our findings point to a previously underappreciated efflux capabilities in Gram-positive bacteria, highlighting the need for better characterization of membrane transporters to clarify their roles in bacterial physiology and drug resistance.

microbiology↗

bioRxiv2026

Reconstitution in a minimal synthetic bacterium reveals distinct Mollicute MreB-based motility systems driven by orthogonal force directions

MreB, a bacterial actin homolog, is widely conserved in bacteria where it functions as a scaffold for a peptidoglycan synthesis. Intriguingly, members of the cell wall-lacking Mollicutes retain multiple mreB genes despite the absence of peptidoglycan. Previous work demonstrated that two MreB isoforms from Spiroplasma can reconstitute swimming motility in the minimal synthetic bacterium syn3B, indicating that MreB was repurposed for motility. Here, we expressed seven MreBs from Haloplasma contractile, an early-diverging Mollicute, in syn3B. Approximately 50% of cells elongated, and 15% exhibited curving and coiling movements that resembled Haloplasma but differed from Spiroplasma motility. Systematic analysis of isoform combinations revealed that movement requires specific MreB pairs, and that additional isoforms enhance motility efficiency. Cryo-electron tomography showed membrane-associated ribbons composed of MreB filaments. Mathematical modeling demonstrated that Haloplasma- and Spiroplasma-type movements can be reproduced by altering only the direction of force generation. Thus, diversification of force orientation within an actin homolog enabled independent evolution of distinct motility systems in Mollicutes.

Synthetic Biology↗

bioRxiv2026

Structure of an active FlhA ring reveals allosteric coupling between substrate specificity and export engine activation

The bacterial flagellar type III secretion system transports structural subunits in a defined order to assemble the flagellum. FlhA is the core component of this nanomachine; its transmembrane domain functions as an ion-driven export engine and its cytoplasmic domain connected by a flexible linker forms a nonameric ring that controls substrate selection. The coupling mechanism of these activities has remained unclear because the transmembrane domain has resisted structural analysis. Here we identify linker deletions that autonomously activate the export gate and capture a functionally active, hook-type protein export state of the entire ring complex. Using cryo-electron microscopy, we determine the structure of the FlhA({Delta}331-347) ring at 2.73 [A] resolution. The structure shows linker-mediated docking of the cytoplasmic ring to the transmembrane ring to stabilize the entire ring while sterically excluding filament-type substrate binding. These findings reveal how the FlhA ring allosterically couples substrate specificity to ion-driven export during flagellar assembly.

microbiology↗

bioRxiv2026

A single conserved residue in FlhA couples export gate activation to substrate specificity switching

FlhA is a core component of the bacterial flagellar type III secretion system that functions as both an export engine and a switch regulator of substrate specificity. How these two processes are mechanistically coupled remains unclear. Here we identify a single conserved residue in FlhA, Arg-391, as a key molecular node that links export gate activation to substrate specificity switching. The R391A substitution blocks the transition from hook-type to filament-type secretion, resulting in polyhooks lacking filaments. An intermolecular salt-bridge formation of Arg-391 with Glu-351 of an adjacent FlhA subunit stabilizes the filament-type FlhA ring. Suppressor mutations in FlhA and the ruler protein FliK restore this interaction, indicating that FliK promotes the intermolecular salt-bridge formation during export switching. Moreover, Arg-391 is required for efficient ATPase-independent activation of the export gate. Together, these findings reveal an electrostatic mechanism by which FlhA coordinates export gate activation and substrate specificity switching during flagellar assembly.

microbiology↗

bioRxiv2026

A region-specific murine intestinal monolayer platform for assessing iron form-dependent transepithelial transport

Iron absorption in the small intestine has classically been described by the duodenal DMT1/FPN1 pathway for inorganic non-heme iron, yet emerging evidence suggests that chemically distinct iron forms may use region-specific routes. Nicotianamine (NA), a plant-derived metal chelator, can form NA-iron (NA-Fe) complexes and has been proposed to support intestinal iron absorption through amino acid transporter pathways. However, direct comparisons of transepithelial transfer of inorganic iron and NA-Fe across defined small intestinal regions under controlled epithelial conditions remain limited. Here, we established region-specific 2D epithelial monolayers derived from duodenal and proximal jejunal crypt organoids from male ICR mice cultured on Transwell inserts. Transcriptomic profiling indicated partial retention of regional identity, and barrier integrity was confirmed by junctional marker localization, transepithelial electrical resistance, and low paracellular permeability. We then examined expression and polarized localization of candidate transporters for inorganic iron (Dmt1/Fpn1) and NA-Fe (Pat1/Lat2). Finally, we quantified transepithelial transport using apical loading of isotope-labeled iron (55Fe) or NA-55Fe and measured radioactivity appearing in the basolateral compartment as the primary readout of transepithelial flux. Basolateral appearance of inorganic 55Fe was comparable between duodenum- and proximal jejunum-derived monolayers, whereas NA-55Fe exhibited significantly greater basolateral appearance in proximal jejunum-derived monolayers. These findings demonstrate that organoid derived, region-specific monolayers provide a tractable epithelial platform to evaluate iron form-dependent, region-specific transepithelial transfer and to enable further mechanistic dissection of NA-Fe transport. NEW & NOTEWORTHYNon-heme iron absorption may depend on iron chemical form and intestinal region, but direct epithelial comparisons are scarce. We established duodenum and proximal jejunum derived murine intestinal organoid monolayers on Transwells and quantified transepithelial flux using isotope-labeled iron. Inorganic 55Fe showed no clear regional difference, whereas NA-55Fe displayed greater basolateral appearance in proximal jejunum-derived monolayers. This platform enables mechanistic studies of NA-iron complex transport.

physiology↗

bioRxiv2026

Active removal of inhibitory components drives the flagellarType III Secretion Specificity Switch

Type III secretion (T3S) systems assemble bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. In both systems, completion of an early assembly intermediate triggers an irreversible switch from early to late substrate secretion. In the flagellar system, this switch is controlled by the secreted molecular ruler FliK acting on the core T3S component FlhB, but the molecular mechanism governing this transition has remained unclear. Here we show that removal of two components, Fluke and the cleaved C-terminal domain of FlhB (FlhBCCD), locks the secretion apparatus in a constitutive late secretion state. In these mutants, secretion specificity no longer requires completion of the hook-basal body or the FliK ruler, indicating that Fluke and FlhBCCD function to maintain the apparatus in early secretion mode. Consistent with this model, synchronized flagellar gene expression experiments reveal that FlhBCCD is retained during early assembly and is lost coincident with hook-basal body completion and activation of {sigma}28-dependent late gene expression of flagellin and chemosensory genes. Structural modeling of the FliK C-terminal switch domain and FlhBCCD supports a mechanism in which secretion of FliK promotes destabilization and ejection of FlhBCCD from the secretion apparatus. Disruption of a folded region within FliK switch domain uncouples secretion from switching, indicating that the timing of FliK unfolding during secretion is critical for activation of the specificity switch. These findings show that secretion specificity switching is driven by FliK-dependent removal of inhibitory components, rather than passive sensing of assembly completion. Significance StatementType III secretion (T3S) systems build complex bacterial nanomachines, including the flagellum and virulence-associated injectisomes, by exporting distinct classes of substrates in a defined temporal order. How these systems switch secretion specificity during assembly has remained a long-standing question. We demonstrate that the flagellar T3S specificity switch requires removal of two inhibitory components that actively maintain the apparatus in an early secretion state. Their FliK-dependent ejection irreversibly triggers the transition to late substrate export, revealing that secretion switching is controlled by active inhibitory regulation rather than passive sensing of assembly completion. These results define a molecular mechanism for hierarchical control in bacterial secretion systems and provide insights likely relevant to other T3S nanomachines.

microbiology↗

bioRxiv2025

Catalytic regulation of CoA transferase by an NAD+-sensing accessory protein and protein acetylation

CoA transferases play essential roles in short-chain fatty acid metabolism by catalyzing the reversible transfer of CoA groups between acyl substrates. However, how their activities are regulated in response to cellular metabolic states remains unclear. Here we identify a dual regulatory mechanism of a CoA transferase from Thermus thermophilus, which is controlled by both interaction with an NAD+-sensing accessory protein and protein acetylation. The enzyme associates with an alanine dehydrogenase-like protein that lacks catalytic activity but binds NAD+ and NADH. Biochemical analyses revealed that inhibition occurs specifically upon NAD+ binding, but not NADH, indicating that the alanine dehydrogenase-like protein senses the intracellular NAD+/NADH ratio. Cryo-electron microscopy structures of the complex of CoA transferase and alanine dehydrogenase-like protein reveal the structural basis of this redox-dependent inhibition. Furthermore, acetylation of the CoA transferase alleviates the inhibition. Since the NAD+/NADH ratio and acetyl-CoA levels reflect the cellular energy and metabolic states, these findings uncover a previously unrecognized regulatory link through which redox sensing and acetylation cooperate to fine-tune the {beta}-oxidation flux.

biochemistry↗

bioRxiv2025

Conversion of an agonistic anti-TNFR2 biparatopic antibody into an antagonist by insertion of peptide linkers into the hinge region

Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or cell internalization, whereas few were reported with smaller immunocomplexes to suppress unwanted signaling. Here, we developed a strategy to induce 1:1 immunocomplex formation to maximize antagonistic function. Various peptide linkers were introduced into the hinge regions of IgG-like agonist BpAbs against tumor necrosis factor receptor 2. Loss of crosslinking activity was observed for one BpAb, allowing the conversion of its function from an agonist to an antagonist. However, cross-linking activity was retained for another agonist BpAb, which binds to a different epitope pair. In a combined analysis of cryo-electron microscopy and coarse-grained molecular dynamics simulations, effect of epitope combination on the stability of 1:1 complexes was observed. These results lead to an understanding of the mechanism and design of BpAbs to adopt a 1:1-binding mode.

biochemistry↗

bioRxiv2025

Structural basis of flagellar rod assembly on the FliPQR protein-export channel

The FliPQR complex constitutes a channel for export of the bacterial flagellar proteins involved in axial structure assembly. It also serves as a template for flagellar rod assembly. A periplasmic gate, formed by the N-terminal -helices of FliP and FliR, remains closed until FliE assembles onto FliP and FliR. The mechanism by which FliE opens the gate and assembles has remained unclear. Here, we present a cryoEM structure of the FliPQR complex in closed form at 3.0 [A] resolution. A {beta}-cap formed by the N-terminal {beta}-strands of FliP and FliR creates a tight seal in the closed gate. Interaction of FliE with FliP and FliR induces a conformational change in FliP and FliR, with their N-terminal -helices move up and outward. Consequently, the N-terminal {beta}-strands of FliP and FliR start opening the periplasmic gate one after another and form a docking site for FliE to initiate rod assembly.

microbiology↗

bioRxiv2025

Chloroplast genome editing of Rubisco boosts photosynthesis and plant growth

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has inherently low catalytic efficiency, making it a key target for photosynthesis and crop improvement. However, introducing mutations to the chloroplast-encoded Rubisco large subunit (rbcL), which contains the enzymes catalytic sites, is technically challenging. In this study, we successfully generated a range of chloroplast-genome-edited Arabidopsis thaliana plants targeting rbcL by a targeted base editor, ptpTALECD. The M309I and D397N substitutions in rbcL resulted in an increased Rubisco catalytic rate (kcat) without any reductions of Rubisco content, thereby enhancing photosynthetic rates and plant growth under both current atmospheric CO2 concentrations (i.e., 381 mol mol-1) and projected future concentrations (i.e., 549 mol mol-1). Cryo-electron microscopy (cryo-EM) structural analysis showed that the M309I and D397N substitutions, although located far from the catalytic site, induce structural alterations in the catalytic (60s) loops. Our findings highlight the potential of Rubisco engineering to improve plant photosynthesis and growth, and underscore the unique opportunities that chloroplast genome editing offers for enhancing photosynthesis and crop productivity and reducing atmospheric CO2 levels in a non-GMO context.

plant biology↗

bioRxiv2024

From Monomers to Oligomers: Structural Mechanism of Receptor-Triggered MyD88 Assembly in Innate Immune Signaling

MyD88 plays a pivotal role in Toll-like receptor (TLR) and interleukin-1 family signaling through its oligomerization upon receptor activation, leading to downstream protein recruitment. The Toll/interleukin-1 receptor domain of MyD88 (TIRMyD88) is responsible for this receptor-mediated oligomerization, but the detailed mechanism involved remains elusive. We investigated the structure of TIRMyD88 oligomers and their interactions with TLRs. Cryoelectron microscopy revealed that tandemly arrayed TIRMyD88 subunits formed an antiparallel double-stranded filament that could further form rings and cylindrical filaments. Moreover, the self-assembly of TIRMyD88 in vitro was markedly accelerated by dimeric rather than monomeric receptor TIRs, possibly reflecting the signal initiation step in vivo. High-speed atomic force microscopy further captured the dynamic processes of oligomerization of TIRMyD88, in addition to its direct interaction with the receptor TIRs. Based on these results, a novel regulatory mechanism of TIRMyD88 oligomerization underlying the signal initiation step was revealed.

biophysics↗

bioRxiv2024

Structural insights into the assembly mechanism of the flagellar MS-ring with three different symmetries

The flagellar basal body MS-ring, formed by 34 FliF subunits, is the core of the flagellar motor as well as the base for flagellar assembly. The MS-ring is also a housing for the flagellar protein export gate complex that is required for construction of the flagellum on the cell surface. A large periplasmic region of FliF contains three ring-building motifs named RBM1, RBM2, and RBM3. RBM3 forms the S-ring and {beta}-collar with C34 symmetry. RBM2 forms the inner core ring of the M-ring with C23 symmetry surrounded by 11 cog-like structures formed by RBM1 and RBM2. However, it remains unknown how FliF assembles to generate these three different symmetries within the MS-ring. Here, we report the two cryoEM structures of the MS-ring formed by FliF co-expressed with FliG and transmembrane export gate proteins. Structural comparison of 33-mer and 34-mer MS-rings revealed that a subtle change in the conformation of RBM3 produces the different rotational symmetries. Combination of cryoEM structural and mutational analyses of the MS-ring with C33 symmetry provides evidence that the well-conserved DQxGxxL motif within a flexible loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3 to form not only 11 cog-like structures just outside the inner core ring with C22 symmetry but also an appropriately sized central pore in the inner core ring to accommodate the export gate complex. IMPORTANCEThe flagellar MS-ring is the core of the flagellar motor and serves not only as an initial template for flagellar assembly but also as a base to accommodate the flagellar protein export complex. The MS-ring is formed by 34 subunits of FliF with two transmembrane helices and a large periplasmic region containing ring-building motifs, RBM1, RBM2, and RBM3. FliF adopts two different conformations in the MS-ring to generate three different rotational symmetries, C34, C23, and C11. However, how FliF assembles to produce these three symmetries remains a mystery. Combination of cryoEM structural and mutational analyses has provided evidence that the well-conserved DQxGxxL motif within a hinge loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3, allowing 23 RBM2 domains of 34 subunits to form the inner core ring with a properly sized central pore to accommodate the flagellar protein export complex.

microbiology↗

bioRxiv2024

Structural basis for the interaction between the bacterial cell division proteins FtsZ and ZapA

Cell division in most bacteria is regulated by the tubulin homolog FtsZ as well as ZapA, a FtsZ-associated protein. However, how FtsZ and ZapA function coordinately has remained elusive. Here we report the cryo-electron microscopy structure of the ZapA-FtsZ complex. The complex forms an asymmetric ladder-like structure, in which the double antiparallel FtsZ protofilament on one side and a single protofilament on the other side are tethered by ZapA tetramers. In the complex, the extensive interactions of FtsZ with ZapA cause a structural change of the FtsZ protofilament, and the formation of the double FtsZ protofilament increases electrostatic repulsion. High-speed atomic force microscopy analysis revealed cooperative interactions of ZapA with FtsZ at a molecular level. Our findings not only provide a structural basis for the interaction between FtsZ and ZapA but also shed light on how ZapA binds to FtsZ protofilaments without disturbing FtsZ dynamics to promote cell division.

molecular biology↗

bioRxiv2024

Dimeric assembly of F1-like ATPase for Mycoplasma gliding motility

Rotary ATPases, including F1Fo- and V/A-ATPases, are molecular motors that exhibit rotational movements for energy conversion1. In the gliding bacterium, Mycoplasma mobile, a dimeric F1-like ATPase forms a chain structure with the glycolytic enzyme, phosphoglycerate kinase (PGK), within the cell2, which is proposed to drive the bacterial gliding motility2-4. However, the mechanisms of force generation and transmission remain unclear. Here, we present a 3.2 [A] resolution structure of the dimeric ATPase complex obtained using electron cryomicroscopy (cryo-EM). Notably, the structure revealed an assembly distinct from that of known dimeric forms of F1Fo-ATPase5, despite containing conserved F1-ATPase structures. The two ATPase units were interconnected by GliD dimers, previously identified as MMOB16202,6. Gli{beta}, a homologue of the F1-ATPase catalytic subunit6, exhibited a specific N-terminal region that incorporates PGK into the complex. Structural conformations of the catalytic subunits, catalytically important residues, and nucleotide-binding pattern of the catalytic sites of the ATPase displayed strong similarities to F1-ATPase, suggesting a rotation based on the rotary catalytic mechanism conserved in rotary ATPases1,7-10. Overall, the cryo-EM structure underscores an evolutionary connection in the rotary ATPases and provides insights into the mechanism through which F1-like ATPase drives bacterial gliding motility.

microbiology↗

bioRxiv2024

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

Nervous necrosis virus (NNV), a non-enveloped betanodavirus, causes neuropathies and retinopathies in farmed fish, damaging aquaculture worldwide. NNV has 60 conspicuous surface protrusions comprising the protrusion domain (P-domain) of its capsid protein. Although NNV protrusions play critical roles in infectivity, the underlying dynamics remain unclear. Our cryogenic electron microscopy (cryo-EM)-derived structures of Dragon grouper (Epinephelus lanceolatus) NNV reveal that the protrusions undergo low-pH-induced compaction and movement. We show that the P-domain is monomeric in solution at a pH germane to infection (7.0). Moreover, nuclear magnetic resonance (NMR) structures reveal a peptide (amino acids 311-330) that adopts a flexible loop to form an open pocket. NMR spectral analysis at pH 5.0 aided by molecular dynamics (MD) simulations show that this loop switches to a {beta}-strand under acidic conditions, eliciting pocket closure and P-domain trimerization, highlighting a unique pH-sensing feature. Our docking analysis revealed the N-terminal moiety of sialic acid inserted into and interacting with conserved residues in the pocket. Additionally, a low-pH-induced conformational change in the linker region via peptide bond isomerization conferred malleability on the protrusions. Our work uncovers the protrusion dynamics of a betanodavirus governing its infectivity through a pH-dependent conformational switching mechanism, providing insights into complex virus-host interactions.

biochemistry↗

bioRxiv2023

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 angstrom

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating cancer cell toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

biochemistry↗