Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII.

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation has been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Observing Dynamic Conformational Changes within the Coiled-Coil Domain of Different Laminin Isoforms Using High-Speed Atomic Force Microscopy.

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques.

An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

Measuring mechanical properties with high-speed atomic force microscopy.

High-speed atomic force microscopy (HS-AFM) is now a widely used technique to study the dynamics of single biomolecules and complex structures. In the past, it has mainly been used to capture surface topography as structural analysis, leading to important discoveries not attainable by other methods. Similar to conventional AFM, the scope of HS-AFM was recently expanded to encompass quantities beyond topography, such as the measurement of mechanical properties. This review delves into various methodologies for assessing mechanical properties, ranging from semi-quantitative approaches to precise force measurements and their corresponding sample responses. We will focus on the application to single proteins such as bridging integrator-1, ion channels such as Piezo1, complex structures such as microtubules and supramolecular fibers. In all these examples, the unique combination of quantifiable force application and high spatiotemporal resolution allows to unravel mechanisms that cannot be investigated by conventional means.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

Removing the parachuting artifact using two-way scanning data in high-speed atomic force microscopy.

The high-speed atomic force microscopy (HS-AFM) is a unique and prominent method to observe structural dynamics of biomolecules at single molecule level at near-physiological condition. To achieve high temporal resolution, the probe tip scans the stage at high speed which can cause the so-called parachuting artifact in the HS-AFM images. Here, we develop a computational method to detect and remove the parachuting artifact in HS-AFM images using the two-way scanning data. To merge the two-way scanning images, we employed a method to infer the piezo hysteresis effect and to align the forward- and backward-scanning images. We then tested our method for HS-AFM videos of actin filaments, molecular chaperone, and duplex DNA. Together, our method can remove the parachuting artifact from the raw HS-AFM video containing two-way scanning data and make the processed video free from the parachuting artifact. The method is general and fast so that it can easily be applied to any HS-AFM videos with two-way scanning data.

Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy.

Cadherin EGF LAG seven-pass G-type receptors (CELSR) cadherins, members of the cadherin superfamily, and adhesion G-protein-coupled receptors, play a vital role in cell-cell adhesion. The mutual binding of the extracellular domains (ectodomains) of CELSR cadherins between cells is crucial for tissue formation, including the establishment of planar cell polarity, which directs the proper patterning of cells. CELSR cadherins possess nine cadherin ectodomains (EC1-EC9) and noncadherin ectodomains. However, the structural and functional mechanisms of the binding mode of CELSR cadherins have not been determined. In this study, we investigated the binding mode of CELSR cadherins using single-molecule fluorescence microscopy, high-speed atomic force microscopy (HS-AFM), and bead aggregation assay. The fluorescence microscopy analysis results indicated that the trans-dimer of the CELSR cadherin constitutes the essential adhesive unit between cells. HS-AFM analysis and bead aggregation assay results demonstrated that EC1-EC8 entirely overlap and twist to form antiparallel dimer conformations and that the binding of EC1-EC4 is sufficient to sustain bead aggregation. The interaction mechanism of CELSR cadherin may elucidate the variation of the binding mechanism within the cadherin superfamily and physiological role of CELSR cadherins in relation to planar cell polarity.

Structure and dynamics of ESCRT-III membrane remodeling proteins by high-speed atomic force microscopy.

Endosomal sorting complex required for transport (ESCRT) proteins assemble on the cytoplasmic leaflet of membranes and remodel them. ESCRT is involved in biological processes where membranes are bent away from the cytosol, constricted, and finally severed, such as in multivesicular body formation (in the endosomal pathway for protein sorting) or abscission during cell division. The ESCRT system is hijacked by enveloped viruses to allow buds of nascent virions to be constricted, severed, and released. ESCRT-III proteins, the most downstream components of the ESCRT system, are monomeric and cytosolic in their autoinhibited conformation. They share a common architecture, a four-helix bundle with a fifth helix that interacts with this bundle to prevent polymerizing. Upon binding to negatively charged membranes, the ESCRT-III components adopt an activated state that allows them to polymerize into filaments and spirals and to interact with the AAA-ATPase Vps4 for polymer remodeling. ESCRT-III has been studied with electron microscopy and fluorescence microscopy; these methods provided invaluable information about ESCRT assembly structures or their dynamics, respectively, but neither approach provides detailed insights into both aspects simultaneously. High-speed atomic force microscopy (HS-AFM) has overcome this shortcoming, providing movies at high spatiotemporal resolution of biomolecular processes, significantly increasing our understanding of ESCRT-III structure and dynamics. Here, we review the contributions of HS-AFM in the analysis of ESCRT-III, focusing on recent developments of nonplanar and deformable HS-AFM supports. We divide the HS-AFM observations into four sequential steps in the ESCRT-III lifecycle: (1) polymerization, (2) morphology, (3) dynamics, and (4) depolymerization.

ATM phosphorylates PP2A subunit A resulting in nuclear export and spatiotemporal regulation of the DNA damage response.

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

Open-source controller for low-cost and high-speed atomic force microscopy imaging of skin corneocyte nanotextures.

High-speed atomic force microscopes (HS-AFMs) with high temporal resolution enable dynamic phenomena to be visualized at nanoscale resolution. However, HS-AFMs are more complex and costlier than conventional AFMs, and particulars of an open-source HS-AFM controller have not been published before. These high entry barriers hinder the popularization of HS-AFMs in both academic and industrial applications. In addition, HS-AFMs generally have a small imaging area that limits the fields of implementation. This study presents an open-source controller that enables a low-cost simplified AFM to achieve a maximum tip-sample velocity of 5,093 µm/s (9.3 s/frame, 512 × 512 pixels), which is nearly 100 times higher than that of the original controller. Moreover, the proposed controller doubles the imaging area to 46.3 × 46.3 µm2 compared to that of the original system. The low-cost HS-AFM can successfully assess the severity of atopic dermatitis (AD) by measuring the nanotexture of human skin corneocytes in constant height DC mode. The open-source controller-based HS-AFM system costs less than $4,000, which provides resource-limited research institutes with affordable access to high-throughput nanoscale imaging to further expand the HS-AFM research community.

Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy.

Classical cadherins play key roles in cell-cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells.

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

In-situ high-speed atomic force microscopy observation of dynamic nanobubbles during water electrolysis.

The nanobubbles (NB) formed on a solid surface has been reported, although the stability in a solution is still discussed. The atomic force microscopy (AFM) has shown the unique shape of the flatted NB, however the dynamic behavior has not investigated yet. Recently, the high scanning speed AFM (HS-AFM) has been developed and applied to the several interfaces. Here, we present in-situ HS-AFM observation during water electrolysis. The hydrogen and oxygen NB evolution on highly oriented pyrolytic graphite (HOPG) electrode are studied. Our video data is the first time to demonstrate the NB nucleation and the growth. The processes are different between both gases. The hydrogen NB grows with active coalescence, while the oxygen one is smaller and irregularly moves on HOPG surface. Using this technique, we will be able to study the NB stability influence by some factors, such as the surface potential and electric capillarity.

Curry Leaf Triggers Cell Death of P. gingivalis with Membrane Blebbing.

Periodontal disease has become a serious public health problem, as indicated by accumulating evidence that periodontal disease is not only a major cause of tooth loss but is also associated with various systemic diseases. The present study assessed the anti-bacterial activities of three herbal products (curry leaf, clove, and cinnamon) against Porphyomonas gingivalis, a keystone pathogen for periodontal diseases. The curry leaf extract (CLE) showed the strongest growth inhibitory activity among them, and the activity was maintained even after extensive heat treatment. Of note, while clove and cinnamon extracts at sub-minimum inhibitory concentrations (sub-MICs) significantly enhanced the biofilm formation of P. gingivalis, CLE at sub-MIC did not have any effect on the biofilm formation. The MIC of CLE against P. gingivalis was higher than those against a wide range of other oral bacterial species. P. gingivalis cells were completely killed within 30 min after treatment with CLE. Spatiotemporal analysis using high-speed atomic force microscopy revealed that CLE immediately triggered aberrant membrane vesicle formation on the bacterial surface. Bacterial membrane potential assay revealed that CLE induced depolarization of the bacterial membrane. Taken together, these findings suggest the mechanism behind early bactericidal activity of CLE and its therapeutic applicability in patients with periodontal diseases.

New Activities of the Nuclear Pore Complexes.

Nuclear pore complexes (NPCs) at the surface of nuclear membranes play a critical role in regulating the transport of both small molecules and macromolecules between the cell nucleus and cytoplasm via their multilayered spiderweb-like central channel. During mitosis, nuclear envelope breakdown leads to the rapid disintegration of NPCs, allowing some NPC proteins to play crucial roles in the kinetochore structure, spindle bipolarity, and centrosome homeostasis. The aberrant functioning of nucleoporins (Nups) and NPCs has been associated with autoimmune diseases, viral infections, neurological diseases, cardiomyopathies, and cancers, especially leukemia. This Special Issue highlights several new contributions to the understanding of NPC proteostasis.

How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System.

The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.

High-speed atomic force microscopy to study pore-forming proteins.

Pore-forming proteins (PFPs) include virulence factors that are produced by many pathogenic bacteria. However, PFPs also comprise non-virulence factors, such as apoptotic Bcl2-like proteins, and also occur in non-pathogenic bacteria and indeed in all kingdoms of life. Pore-forming proteins are an ancient class of proteins, which are tremendously powerful in damaging cell membranes. In general, upon binding to lipid membranes, they convert from the soluble monomeric form into an oligomeric state, and then undergo a dramatic conformational change to form transmembrane pores. Thus, PFPs render the plasma membrane of their target cells permeable to solutes, potentially leading to cell death, or to more subtle manipulations of cellular functions. Recent cryo-EM and X-ray crystallography studies revealed high-resolution structures of several PFPs in their pre-pore and pore states, however many aspects regarding the cues that induce pore formation, the pre-pore to pore conformational transition, the mechanism of membrane permeation and associated dynamics are still less well understood, and direct visualization of the dynamics of these transitions are missing. Using high-speed atomic force microscopy (HS-AFM), the kinetics of oligomerization and the pre-pore to pore transition dynamics of various PFPs, such as Listeriolysin O (LLO), lysenin, and Perforin-2 (PFN2), could be studied. These studies revealed that LLO does not form pores of regular shape or size, but rather forms membrane inserted arcs that propagate and damage lipid membranes as lineactants. In contrast, lysenin forms stable pre-pore and pore nonameric rings and HS-AFM allowed to study their diffusion on and the pH-dependent insertion into the membrane. Similarly, PFN2 underwent pre-pore to pore transition upon acidification. The openness of the HS-AFM system allowed the transition to be imaged in real time and revealed that all observed molecules transitioned into the pore state within 3s. In this chapter, we detail protocols to prepare lipids, form supported lipid bilayers, and provide guidelines for real-time, real-space HS-AFM observations of PFPs in action.

Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal P-stalk by high-speed AFM.

In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0•(aP1•aP1)3 (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.

Spatiotemporally tracking of nano-biofilaments inside the nuclear pore complex core.

Nuclear pore complex (NPC) is a gating nanomachine with a central selective barrier composed mainly of Nups, which contain intrinsically disordered (non-structured) regions (IDRs) with phenylalanine-glycine (FG) motifs (FG-NUPs). The NPC central FG network dynamics is poorly understood, as FG-NUPs liquid-liquid phase separation (LLPS) have evaded structural characterization. Moreover, the working mechanism of single FG-NUP-biofilaments residing at the central lumen is unknown. In general, flexible biofilaments are expected to be tangled and knotted during their motion and interaction. However, filament knotting visualization in real-time and space has yet to be visualized at the nanoscale. Here, we report a spatiotemporally tracking method for FG-NUP organization with nanoscale resolution, unveiling FG-NUP conformation in NPCs of colorectal cells and organoids at timescales of ~150 ms using high-speed atomic force microscopy (HS-AFM). Tracking of FG-NUP single filaments revealed that single filaments have a heterogeneous thickness in normal and cancer models which in turn affected the filament rotation and motion. Notably, FG-NUPs are overexpressed in various cancers. Using the FG-NUP inhibitor, trans-1,2-cyclohexanediol, we found that central plug size was significantly reduced and incompletely reversible back to filamentous structures in aggressive colon cancer cells and organoids. These data showed a model of FG-NUPs reversible self-assembly devolving into the central plug partial biogenesis. Taken together, HS-AFM enabled the tracking and manipulation of single filaments of native FG-NUPs which has remained evasive for decades.

High-speed atomic force microscopy directly visualizes conformational dynamics of the HIV Vif protein in complex with three host proteins.

Vif (viral infectivity factor) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to disrupt the antiviral activity of host APOBEC3 (apolipoprotein B mRNA-editing enzyme catalytic subunit 3) proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and core-binding factor subunit ß, forming a four-protein complex called VCBC. The structure of VCBC-Cullin5 has recently been solved by X-ray crystallography, and, using molecular dynamics simulations, the dynamics of VCBC have been characterized. Here, we applied time-lapse high-speed atomic force microscopy to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of this complex, which we identified as the triangle, dumbbell, and globular structures. Moreover, we characterized the dynamics of each of these structures. Our data revealed the very dynamic behavior of all of them, with the triangle and dumbbell structures being the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and may support efforts to improve HIV treatment, because Vif is essential for virus survival in the cell.