Structural maintenance of chromosomes (SMC) complexes, including cohesin and condensin, are ring-shaped ATPases that organize chromosome architecture by holding DNA segments together. However, the dynamic mechanisms underlying their DNA binding and processing remain unclear. Using high-speed atomic force microscopy, we directly visualized the eukaryotic Smc5/6 complex on DNA at submolecular resolution. Smc5/6 adopted an “I”-shaped conformation with closely aligned SMC arms and transitioned to an “O”-shaped open ring upon ATP binding. ATP-bound Smc5/6 stably associated with DNA through its ATPase head domains, whereas ATP hydrolysis promoted DNA entrapment within the SMC compartment and repositioning near the hinge. Smc5/6 also tethered two DNA segments together and stabilized twisted DNA structures, leading to DNA compaction. Our findings provide a visual demonstration of how an SMC complex employs its ring architecture to facilitate distinct DNA binding modes, deepening our understanding of the ATP-dependent dynamic transactions of Smc5/6 during DNA binding. SMC complexes organize higher-order genome architecture through their unique DNA-binding activities. Using HS-AFM the authors reveal the submolecular dynamics of Smc5/6 on DNA, providing mechanistic insight into this ATP-dependent interaction.

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. Here we investigate the structure of TIRMyD88 oligomers and their interactions with TLRs. Cryoelectron microscopy reveals that tandemly arrayed TIRMyD88 subunits form an antiparallel double-stranded filament that can further form rings and cylindrical filaments. Moreover, the self-assembly of TIRMyD88 in vitro is markedly accelerated by dimeric rather than monomeric receptor TIRs, possibly reflecting the signal initiation step in vivo. High-speed atomic force microscopy further captures the dynamic processes of oligomerization of TIRMyD88, in addition to its direct interaction with the receptor TIRs. These results reveal a regulatory mechanism of TIRMyD88 oligomerization underlying the signal initiation step.

Recent technical advances in atomic force microscopy (AFM) have led to the development of high-speed AFM (HS-AFM), which enables video-rate imaging and direct visualization of the nanoscale dynamics of biomolecules functioning in solution. Because HS-AFM rapidly generates large datasets, dedicated software is essential for efficient processing, organization, and analysis. Recent improvements in imaging speed have further increased data throughput, underscoring the need for faster analysis software with improved usability. To address this need, we developed Ultrafast Microscopy Exploration (UMEX) Viewer, a software suite for HS-AFM data analysis. UMEX Viewer provides an integrated environment for HS-AFM data handling, including image processing, drift correction, image and movie export, data management, and quantitative analysis. These capabilities have enabled the software to be applied to a wide range of recent studies. In this article, we describe the core functions of UMEX Viewer and demonstrate its practical applications for HS-AFM data analysis aimed at elucidating biological functions.

Atomic force microscopy (AFM) is a versatile nanoscale imaging technique. Since its force detection accuracy and spatiotemporal resolution are fundamentally limited by the minimum detectable force (MDF) arising from system noise, a deep understanding of MDF is essential for both instrumentation and applications. In particular, elucidating the imaging conditions that minimize the MDF is crucial for observing fragile supramolecular complexes. However, the theoretical MDF of amplitude-modulation (AM) AFM has received little attention, and its formulations remain inconsistent. Specifically, although we recently clarified the strong dependence of force sensitivity on the cantilever's driving frequency, the optimal excitation frequency from the MDF perspective remains unknown. Here, we present an exact solution for the MDF of AM-AFM that accounts for excitation-frequency-dependent noise and force conversion. Our results show that MDF is strongly governed by the driving frequency and the cantilever Q-factor. In the repulsive regime, driving above the resonance peak frequency causes a sharp MDF increase, exceeding that of other dynamic modes by over an order of magnitude, whereas lower-side-slope excitation yields only similar to 1.41-1.73 times larger MDF; similar behavior is observed in the attractive regime. These findings resolve a long-standing inconsistency in the MDF of AM-AFM and demonstrate that slope excitation, rather than resonance excitation, enables the detection of several-fold weaker forces, leading to more accurate force estimation and visualization of previously unobservable fragile molecules.

Stress granules (SGs) are stress-induced ribonucleoprotein condensates assembled around untranslated mRNAs and RNA-binding proteins. G3BP1 is a central regulator of SG formation, yet the molecular events that initiate G3BP1-mediated condensation remain poorly understood. Current models propose that condensation is initiated by RNA-RNA interactions, G3BP1 self-association, or RNA-dependent assembly of G3BP1 into higher-order networks. To define the earliest steps of condensate formation, we employed high-speed atomic force microscopy (HS-AFM) to monitor G3BP1-RNA assembly at the nanometer scale. HS-AFM revealed that G3BP1 first associates with RNA to form discrete nascent assemblies that progressively recruit additional RNA and G3BP1 molecules. These assemblies subsequently grow into higher-order RNA-protein condensates through stepwise assembly. Together, these observations identify RNA-bound G3BP1 assemblies as the initiating structures of condensate formation and provide a framework for understanding the early stages of stress granule assembly.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) is highly enriched in dendritic spines at concentrations comparable to those of cytoskeletal proteins and plays a central role in synaptic plasticity. During long-term potentiation, CaMKIIα further accumulates in spines. However, the mechanisms governing its higher-order organization remain poorly understood. Here, we use high-speed atomic force microscopy to visualize interholoenzyme interaction of CaMKIIα at mesoscopic scales (5 to 500 nanometers). Under freely diffusible conditions, CaMKIIα holoenzymes do not form stable clusters. In contrast, when spatially confined, they assemble into chain-like clusters mediated by kinase-domain interactions. These clusters expand upon activation, concomitant with the dissociation of the regulatory segment. Notably, the CaMKIIα Pro 212 →Leu (P212L) mutant associated with neurodevelopmental disorders forms extensive clusters even in the basal state. Together, our findings demonstrate that CaMKIIα-CaMKIIα interactions drive mesoscale cluster formation and that precise regulation of cluster size and activation-dependent growth might be critical for synaptic signaling.

Synovial sarcoma is driven by the SS18–SSX fusion oncoprotein, in which the SSX C-terminal region replaces SMARCB1 in the BAF chromatin remodeling complex. However, the structural basis by which SSX displaces SMARCB1, and the potential functional differences between SSX1 and SSX2 have remained unclear. Here, we determined cryo-electron microscopy structures of SSX1RD– and SSX2RD–unmodified nucleosome complexes at a resolution of 2.4 Å and 2.5 Å, respectively. Both structures revealed conserved interactions mediated by the SSX RLR motif with the acidic patch, a cation–π interaction between W164 and R167, and insertion of Y177 into a hydrophobic pocket of H2A, indicating that SSX1RD and SSX2RD are functionally interchangeable in nucleosome binding. Molecular dynamics simulations and binding free-energy analyses showed that SSXRD binds the acidic patch more strongly than SMARCB1, explaining how SSX fusion proteins displace SMARCB1 during BAF assembly. Complementary circular dichroism and NMR spectroscopy confirmed that SSX1(111–188) and SSX2(111–188) are intrinsically disordered regions capable of weak nucleosome binding and nuclear condensates formation. Together, these findings establish a mechanistic and structural framework for understanding SSX-driven chromatin remodeling, offering a basis for therapeutic strategies targeting SSX-mediated BAF dysregulation in synovial sarcoma.

Afamin is a serum glycoprotein that stabilizes and transports hydrophobic signaling protein Wnt3a, but the structural basis of this function has remained unclear. Here, we combined high-speed atomic force microscopy and atomistic molecular modeling to investigate this complex in solution. Our analysis revealed that afamin exhibits the motion of two globular domains opening and closing through the hinge regions. An Afamin/Wnt3a complex adopts two interconvertible conformations: a symmetric conformation, where Wnt3a positions at the center between the two globular domains of afamin, and an asymmetric conformation, where Wnt3a positions on the N-terminal-side domain of the afamin. Binding to Wnt3a reduced the intrinsic flexibility of afamin. Additionally, cell-based binding experiments demonstrated that stable Wnt3a association requires the intact architecture of afamin containing an undisturbed hydrophobic pocket region. Together, these findings suggest that the Afamin/Wnt3a complex adopts its dynamic states, with the lipid moiety of Wnt3a embedded in the hydrophobic pocket of afamin.

ABSTRACT Although the maturation state of dengue virus (DENV) particles is a key determinant of their infectivity, maturation is unusually inefficient. Fully mature and immature DENV particles are well-studied; however, little is known about partially mature particles. Moreover, single-particle structural dynamics and nanomechanical properties are unknown. Here, we observe wildtype and immature DENV particles using a single-particle approach combining high-speed AFM (HS-AFM) and 3D force mapping (3DFM). HS-AFM shows that the conformations of each morphotype are heterogeneous and dynamic in liquid, particularly partially mature virions. Tracking immature prM–E spikes elucidates their dynamic movements, which show intraviral variation and constrained independence. 3DFM measurements suggest that internal DENV structure is also heterogeneous and undergoes maturation-dependent changes, with the nucleocapsid core not occupying the full internal volume of immature virions. This approach complements current structural virology techniques and adds a new dimension to our understanding of the structural properties of viruses.

EML4-ALK is a key oncogenic driver in lung cancer, but variant-specific chemoresistance limits the efficacy of current ALK inhibitors. Because the N-terminus contains an intrinsically disordered region (IDR), how ALK inhibitors affect the structure and dynamics of full-length EML4-ALK remains unclear. Here, using high-speed atomic force microscopy (HS-AFM), we visualize the overall structures of three full-length EML4-ALK variants (v1, v3, and v5) at the single-molecule level. We identified a transient globular subdomain (residues 191-217) within the v3 IDR that contributes to distinct oligomerization patterns. Notably, ALK inhibitors compact the IDR subdomain and reduce oligomerization, whereas this effect is abolished by the resistance mutation ALKG1202R, suggesting that ALK inhibitors not only inhibit kinase activity but also modulate IDR dynamics. Our study reveals a structural basis linking IDR dynamics to variant-specific oligomerization in EML4-ALK, offering further insights into the regulation of oncogenic signaling and the development of targeted therapies.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in dendritic spines at concentrations comparable to those of cytoskeletal proteins and plays a central role in synaptic plasticity. During long-term potentiation (LTP), CaMKIIα further accumulates in spines. However, the mechanisms governing its higher-order organization remain poorly understood. Here, we use high-speed atomic force microscopy to visualize inter-holoenzyme interaction of CaMKIIα at mesoscopic scales (5–500 nm). Under freely diffusible conditions, CaMKIIα holoenzymes do not form stable clusters. In contrast, when spatially confined, they assemble into worm-like chain clusters mediated by kinase-domain interactions. These clusters expand upon activation, concomitant with the dissociation of the regulatory segment. Notably, the CaMKIIα P212L mutant associated with neurodevelopmental disorders, forms extensive clusters even in the basal state. Together, our findings demonstrate that CaMKIIα-CaMKIIα interactions drive mesoscale cluster formation and that precise regulation of cluster size and activation-dependent growth might be critical for synaptic signaling. ### Competing Interest Statement The authors have declared no competing interest. the World Premier International Research Center Initiative (WPI), MEXT, Japan JSPS KAKENHI, JP24K21942, JP25H00972, JP22H04926 the Mochida Memorial Foundation for Medical and Pharmaceutical Research Uehara Memorial Foundation, https://ror.org/00gc20a07 Naito Foundation, https://ror.org/02s016q17 JST CREST, JPMJCR1762 JST SPRING, JPMJSP2135 JST ERATO, JPMJER2403

Smooth muscle myosin light chain phosphatase (MLCP), composed of a catalytic subunit PP1c and large and small noncatalytic subunits (MYPT1 and M20, respectively), is a key mediator of Rho-associated coiled-coil-containing kinase (RhoA-ROCK) signaling in cytoskeletal regulation. Phosphorylation of MYPT1 at Thr696 and Thr853 inhibits MLCP activity and augments MLC phosphorylation and smooth muscle contraction, whereas the functions of M20 remain undefined. To elucidate the functional significance of M20 in MLCP regulation, the present study compared the biochemical and structural properties of recombinant MLCP trimer (PP1c, MYPT1, and M20) and dimer (PP1c and MYPT1) complexes. While the rate of MYPT1 phosphorylation at Thr696 and Thr853 by ROCK2 in the presence of calyculin A was indistinguishable between the trimer and dimer complexes, the subsequent autodephosphorylation initiated by ROCK2 inhibition was significantly slower in the trimer. In the absence of calyculin A, phosphorylation in the trimer was greater than in the dimer. A dimer containing C-terminally truncated MYPT1 (Δ931 to 1030) mimicked trimer properties. Pull-down assays demonstrated the interactions of MYPT1-M20 and MYPT1-MYPT1, which were abolished by MYPT1 (Δ931 to 1030). High-speed atomic force microscopy revealed a superdimer complex consisting of two MLCP dimers tethered to the C-terminal region, whereas the trimer retained a single entity. These findings reveal a function of M20 that augments the inhibitory phosphorylation of MYPT1 by preventing superdimer formation mediated by the C-terminal region of MYPT1 and suppressing autodephosphorylation. Knockdown of M20 decreased the basal phosphorylation of MYPT1. M20 thus maintains MLCP in the inhibited state at basal conditions.

Atomic force microscopy (AFM) enables nanoscale characterization and has been widely applied to a broad range of systems. Over the past two decades, advances in high-speed AFM have enabled not only the imaging of static structures but also the direct observation of nanoscale dynamics in real time. However, because the tip or sample is typically scanned using piezoelectric actuators, nonlinearities in their response to the input signal can introduce image-scaling errors of up to 20-30%. Consequently, there is a strong demand for a method to correct piezoelectric nonlinearity that can reliably support quantitative dynamic structural measurements. Here, we propose a simple software-based feedforward method to generate scan waveforms that can be readily implemented. We identify four distinct sources of positioning error in piezo scanners and demonstrate that these errors can be compensated, achieving an order-of-magnitude improvement in positioning accuracy compared with uncompensated operation. Because the proposed method is software-based and requires no additional hardware, it preserves imaging speed and is well suited for high-speed AFM. It is also compatible with a wide range of AFM and other scanning probe systems.

In this Comment, we direct attention to initial efforts to establish a high-quality databank of atomic force microscopy (AFM) data: bioAFM-DB. We outline the state of this endeavor, its challenges, and potential courses of action.

Cells achieve metabolic precision by assembling enzymes into dynamic complexes, but the regulatory mechanism of these metabolons is unclear. Here we characterize the chalcone synthase (CHS)-chalcone isomerase-like protein (CHIL) complex, a key component of flavonoid metabolons. While crystallography provides a static view, our analyses reveal that CHS undergoes rapid, reversible binding cycles with CHIL that regulate catalysis in real time. CHIL removes coenzyme A, an inhibitor of Claisen cyclization, transiently reshapes the CHS active site and guides the tetraketide intermediate towards productive cyclization, thereby suppressing derailment by-products. These findings demonstrate a previously unproven and generalizable regulatory effect in metabolons: guided active-site tuning via transient enzyme association. This concept enhances our understanding of metabolon function and opens avenues for synthetic biology and metabolic engineering.

Cyclase-associated protein (CAP) binds to both actin monomers and filaments and regulates multiple aspects of the actin dynamics including polymerization and depolymerization. CAP has been isolated from multiple species as a stable equimolar complex with actin monomer. However, functional significance of the CAP-actin complex is unknown. We previously demonstrated that native Xenopus cyclase-associated protein 1 (XCAP1) forms a 4:4 complex with actin. Here, we characterized how actin-free XCAP1 and the XCAP1-actin complex interact with actin filaments using high-speed atomic force microscopy and found that XCAP1-bound actin monomers influence dwell time and positional preference of XCAP1 on actin filaments. Actin-free XCAP1 bound to actin filaments transiently with a dwell time of ∼0.2 seconds. The XCAP1-actin complex also bound to actin filaments transiently but with a 3- to 5-fold longer dwell time than actin-free XCAP1. Actin-free XCAP1 bound to both side and ends of actin filaments with moderate preference to ends. However, the XCAP1-actin complex bound preferentially to the side of actin filaments. These results indicate that binding of actin monomers to XCAP1 affects its binding modes to actin filaments, suggesting that this might be a novel mechanism to regulate the effects of CAP on actin filaments.

Biomolecular condensation is a fundamental mechanism for organizing intracellular components without membranes. While RNA self-assembly has been implicated in condensate formation, it remains unclear how RNA condensation translates into distinct physical behaviors at the nanoscale. Utilizing high-speed atomic force microscopy (HS-AFM), this study characterizes RNA condensation dynamics with nanoscale resolution. Imaging captured the transition from individual RNA folding to intermolecular clustering, ultimately leading to progressive condensate assembly. Beyond morphological description, condensate behavior was further examined through fusion dynamics and mechanical response. Post-fusion shape evolution quantified how merged condensates recover circular morphology over time, providing a dynamic readout of material behavior. Nanomechanical properties were independently assessed through force-curve measurements. Together, these analyses consistently distinguish the liquid-like poly A condensates from solid-like condensates of total RNA. Taking together, these findings directly link RNA folding dynamics, condensate assembly, and emergent physical properties, establishing a quantitative framework for defining condensate material states at the nanoscale.

Abstract High-speed atomic force microscopy (HS-AFM) enables visualization of biomolecular dynamics in solution with high spatiotemporal resolution but requires substrates that support stable adsorption without perturbing their structure or dynamics. Mica is widely used as an HS-AFM substrate because it provides an atomically flat surface. Several methods have been proposed to functionalize mica surfaces, but they are not suitable for all biomolecules. Here, we expand the HS-AFM substrate toolbox by introducing methyl- and carboxyl (COOH)-functionalized mica surfaces based on a common silanization strategy. In particular, a COOH-functionalized mica substrate was prepared via a mild thiol–maleimide coupling reaction, enabling efficient surface modification under aqueous conditions. Using proteins with different charges and shapes, we evaluated adsorption and diffusion behaviors on these substrates by HS-AFM. Distinct differences in molecular mobility and binding were observed depending on surface properties. These results provide practical guidelines for substrate selection and broaden the applicability of HS-AFM to diverse biomolecular systems.

Extracellular antimicrobial resistance genes (exARGs) and viruses in wastewater and aquatic environments pose significant public health risks. Microfiltration (MF) membranes have limited effectiveness in removing exARGs and viruses due to their nanoscale size, and removal primarily relies on adsorption onto membrane surfaces. This study aimed to (i) evaluate the adsorption of exARGs and viruses on different membrane materials and (ii) clarify the effect of ionic conditions on their adsorption. A recombinant plasmid (model exARG), nonenveloped virus MS2, and enveloped virus phi 6 were spiked into salt solutions to assess adsorption onto PES and PVDF membranes. As a result, phi 6 exhibited greater adsorption than MS2, which was attributed to its higher hydrophobicity. PES membranes showed significantly higher MS2 adsorption compared to PVDF. According to the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory, phi 6 exhibited less repulsion than MS2 and exARGs, supporting its enhanced adsorption. Furthermore, the presence of Ca2+ enhanced exARG adsorption by 0.79 log on PES membranes. These findings suggest that optimizing membrane material selection and ionic conditions can enhance the adsorptive removal of exARGs and viruses, offering a strategy to improve the MF membrane performance in water treatment applications.

High-speed atomic force microscopy (HS-AFM) experiments allow direct observation of biomolecular dynamics at the single-molecule level, acquiring a large amount of topographic imaging data that visualizes changes in the molecular surface during functional activity over an extended period of time. Since images have no atomistic resolution, a major challenge has been to develop post-experimental computational methods to infer atomistic information from measurements. The recently developed NMFF-AFM flexible fitting method provides a computationally efficient approach promising to infer atomistic-precision models of conformational dynamics from resolution-limited AFM imaging data. We report the software integration of this method into the well-established BioAFMviewer platform and demonstrate its first applications to experimental HS-AFM imaging data. To facilitate applications, we developed a direct workflow from raw experimental AFM data to the visualization and analysis of fitting results. The presented applications to experimental data of a single protein domain, a protein complex, and a megadalton-sized protein filament demonstrate the versatility of NMFF-AFM modeling to reproduce large-amplitude conformational motions of biomolecular dynamics from HS-AFM imaging. As a first step toward automated large-scale analysis of AFM imaging data, we furthermore demonstrate reconstruction of an atomistic molecular movie of protein dynamics, involving large-amplitude conformational transitions, from a measured HS-AFM movie sequence. Implementation of flexible fitting within the stand-alone user-friendly interactive BioAFMviewer software provides the opportunity for a broad range of applications to facilitate the understanding of resolution-limited HS-AFM measurements.