Mechanical Stress Decreases the Amplitude of Twisting and Bending Fluctuations of Actin Filaments.

A variety of biological roles of mechanical forces have been proposed in cell biology, such as cell signaling pathways for survival, development, growth, and differentiation. Mechanical forces alter the mechanical conditions within cells and their environment, which strongly influences the reorganization of the actin cytoskeleton. Single-molecule imaging studies of actin filaments have led to the hypothesis that the actin filament acts as a mechanosensor; e.g., increases in actin filament tension alter their conformation and affinity for regulatory proteins. However, our understanding of the molecular mechanisms underlying how tension modulates the mechanical behavior of a single actin filament is still incomplete. In this study, a direct measurement of the twisting and bending of a fluorescently labeled single actin filament under different tension levels by force application (0.8-3.4 pN) was performed using single-molecule fluorescence polarization (SMFP) microscopy. The results showed that the amplitude of twisting and bending fluctuations of a single actin filament decreased with increasing tension. Electron micrograph analysis of tensed filaments also revealed that the fluctuations in the crossover length of actin filaments decreased with increasing filament tension. Possible molecular mechanisms underlying these results involving the binding of actin-binding proteins, such as cofilin, to the filament are discussed.

[Method for Measuring Noise Power Spectrum Depending on X-ray Tube Angle in Spiral Orbit of Helical CT Scan].

PURPOSE: The noise power spectrum (NPS) in computed tomography (CT) images potentially varies with the X-ray tube angle in a spiral orbit of the helical scan. The purpose of this study was to propose a method for measuring the NPS for each angle of the X-ray tube. METHODS: Images of the water phantom were acquired using a helical scan. As a conventional method, we measured the two-dimensional (2D) NPS from each image and averaged them; the obtained 2D-NPS was referred to as NPSconventional. In the proposed method, we made the X-ray tube angle θ (0°≤θ<360°) to correspond to the image according to each slice position of the images that located within the travel distance of the CT scan table per 360° rotation of the X-ray tube. We obtained the 2D-NPS from each image and assigned the θ (0°, 30°, 60°, 90°, 120°, 150°, 180°); the obtained 2D-NPS was referred to as NPSsθ. The NPSsθ was compared to the NPSconventional. Also, we investigated the dependency of the NPSsθ on the θ. RESULTS: The NPSconventional was found to be isotropic, and in contrast, the NPSsθ was anisotropic. The NPSsθ showed a continuously rotational change while increasing the θ. There was an excellent correlation (R2>0.999) between the rotation angle of NPSθ and the θ. CONCLUSION: The proposed method was demonstrated to be effective for evaluating anisotropic noise characteristics depending on the X-ray tube angle.

Method for measuring noise-power spectrum independent of the effect of extracting the region of interest from a noise image.

This study aimed to evaluate the impact of region of interest (ROI) size on noise-power spectrum (NPS) measurement in computed tomography (CT) images and to propose a novel method for measuring NPS independent of ROI size. The NPS was measured using the conventional method with an ROI of size P × P pixels in a uniform region in the CT image; the NPS is referred to as NPSR=P. NPSsR=256, 128, 64, 32, 16, and 8 were obtained and compared to assess their dependency on ROI size. In the proposed method, the true NPS was numerically modeled as an NPSmodel, with adjustable parameters, and a noise image with the property of the NPSmodel was generated. From the generated noise image, the NPS was measured using the conventional method with a P × P pixel ROI size; the obtained NPS was referred to as NPS'R=P. The adjustable parameters of the NPSmodel were optimized such that NPS'R=P was most similar to NPSR=P. When NPS'R=P was almost equivalent to NPSR=P, the NPSmodel was considered the true NPS. NPSsR=256, 128, 64, 32, 16, and 8 obtained using the conventional method were dependent on the ROI size. Conversely, the NPSs (optimized NPSsmodel) measured using the proposed method were not dependent on the ROI size, even when a much smaller ROI (P = 16 or 8) was used. The proposed method for NPS measurement was confirmed to be precise, independent of the ROI size, and useful for measuring local NPSs using a small ROI.

Mutagenic analysis of actin reveals the mechanism of His161 flipping that triggers ATP hydrolysis.

The dynamic assembly of actin is controlled by the hydrolysis of ATP, bound to the center of the molecule. Upon polymerization, actin undergoes a conformational change from the monomeric G-form to the fibrous F-form, which is associated with the flipping of the side chain of His161 toward ATP. His161 flipping from the gauche-minus to gauche-plus conformation leads to a rearrangement of the active site water molecules, including ATP attacking water (W1), into an orientation capable of hydrolysis. We previously showed that by using a human cardiac muscle α-actin expression system, mutations in the Pro-rich loop residues (A108G and P109A) and in a residue that was hydrogen-bonded to W1 (Q137A) affect the rate of polymerization and ATP hydrolysis. Here, we report the crystal structures of the three mutant actins bound to AMPPNP or ADP-Pi determined at a resolution of 1.35-1.55 Å, which are stabilized in the F-form conformation with the aid of the fragmin F1 domain. In A108G, His161 remained non-flipped despite the global actin conformation adopting the F-form, demonstrating that the side chain of His161 is flipped to avoid a steric clash with the methyl group of A108. Because of the non-flipped His161, W1 was located away from ATP, similar to G-actin, which was accompanied by incomplete hydrolysis. In P109A, the absence of the bulky proline ring allowed His161 to be positioned near the Pro-rich loop, with a minor influence on ATPase activity. In Q137A, two water molecules replaced the side-chain oxygen and nitrogen of Gln137 almost exactly at their positions; consequently, the active site structure, including the W1 position, is essentially conserved. This seemingly contradictory observation to the reported low ATPase activity of the Q137A filament could be attributed to a high fluctuation of the active site water. Together, our results suggest that the elaborate structural design of the active site residues ensures the precise control of the ATPase activity of actin.

Cryo-EM structure of the Agrobacterium tumefaciens T4SS-associated T-pilus reveals stoichiometric protein-phospholipid assembly.

Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer.

ATP-dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. This protein polymerizes into an antiparallel double-stranded filament that determines cell shape by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five MreB homologous (SpeMreB1-5) that probably contribute to swimming motility. Here, we investigated the structure, ATPase activity and polymerization dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerized into a double-stranded filament with possible antiparallel polarity, while SpeMreB5 formed sheets which contained the antiparallel filament, upon nucleotide binding. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our SpeMreB3 crystal structures and analyses of SpeMreB3 and SpeMreB5 variants showed that the amino acid motif probably plays a role in eliminating a nucleophilic water proton during ATP hydrolysis. Sedimentation assays suggest that SpeMreB3 has a lower polymerization activity than SpeMreB5, though their polymerization dynamics are qualitatively similar to those of other actin superfamily proteins, in which pre-ATP hydrolysis and post-Pi release states are unfavourable for them to remain as filaments.

Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

[A Study of Longitudinal NPS Measurement in CT Images Based on the Central Cross-section Theorem].

PURPOSE: Various approaches in noise power spectrum (NPS) analysis are currently used for measuring a patient's longitudinal (z-direction) NPS from three-dimensional (3D) CT volume data. The purpose of this study was to clarify the relationship between those NPSs and 3D-NPS based on the central slice theorem. METHODS: We defined the 3D-NPS(fx, fy, fz) that was calculated by 3D Fourier transform (FT) from 3D noise data (3D-Noise(x, y, z), x-y scan plane). Here, fx, fy and fz are spatial frequencies corresponding to the axes of x, y and z, respectively. Based on the central slice theorem, we described three relationships as follows. (1) The fz-directional NPS calculated from the 3D-Noise(x=0, y=0, z) is equal to the profile obtained by projecting 3D-NPS(fx, fy, fz) in fx- and fy-directions. (2) The fz-directional NPS calculated from the profile obtained by projecting 3D-Noise(x=0, y, z) in the y-direction is equal to the profile at fy=0 in the data obtained by projecting 3D-NPS(fx, fy, fz) in the fx-direction. (3) The fz-directional NPS calculated from the profile obtained by projecting 3D-Noise(x, y, z) in x and y-directions is equal to the profile of 3D-NPS(fx=0, fy=0, fz). To verify them, we compared the NPSs measured from actual 3D noise data that were obtained using a cylindrical water phantom. RESULTS: In each relationship (1)-(3), the fz-directional NPS matched the profile obtained from the 3D-NPS(fx, fy, fz). CONCLUSION: Based on the central slice theorem, we clarified the relationships between fz-directional NPSs and 3D-NPS. We should understand them and then consider which method should be used for fz-directional NPS measurement.

[An Easy-to-use Method for CT Image Simulation with Parameter Optimization Using a Water Phantom].

PURPOSE: We developed an easy-to-use method to generate computed tomography (CT) images that simulate the images obtained when using an actual scanner. METHODS: The developed method generates images by simulating the data acquisition and image reconstruction processes of a scanner from a linear attenuation coefficient map of an object numerically generated. This approach is similar to general image simulation methods. However, we introduced adjustable parameters for the CT data acquisition process, for example, parameters related to X-ray attenuation in the anode of the X-ray tube and the bowtie filter. These parameters were optimized in advance by minimizing the difference between the simulated and measured images of a water phantom. To verify the validity of the developed method, a simulated image was generated for a torso phantom and then compared with the measured image of the phantom obtained using the scanner. RESULTS: The simulated and measured images of the torso phantom were in good agreement. The spatial resolution and noise characteristics of these two images were also comparable, further indicating the accuracy of the developed method. CONCLUSION: In the existing methods, various information/data related to an actual scanner, including difficult-to-acquire ones, were essential for image simulation. In the developed method, instead of determining the difficult-to-acquire information/data, we introduced adjustable parameters. Therefore, the developed method was easier to use than the existing methods.

The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes.

Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

[A Study of 3D-NPS Analysis in CT Images Based on the Central Cross-section Theorem].

PURPOSE: The noise power spectrum (NPS) of a CT scanner is commonly measured from a single noise image. However, since CT images are three-dimensional (3D) volume data, they have 3D noise characteristics (3D-NPS). In this study, we clarify the relationship among NPSs measured by various approaches in NPS analysis based on the central slice theorem. Its validity is verified by the NPS measurements using actual 3D noise data. METHODS: We defined the NPSz-projection(fx, fy) that was calculated by the 2D Fourier transform (FT) from the 2D projection of 3D noise data in the patient longitudinal direction, the 3D-NPS(fx, fy, fz) that was calculated by the 3D-FT from the 3D noise data, and the 2D-NPS(fx, fy) that was calculated by the 2D-FT from a single noise image; fx, fy, and fz are spatial frequencies corresponding to the axes of x, y, and z in the reconstructed CT volume, respectively. Based on the central slice theorem, we described that the NPSz-projection(fx, fy=0) was equal to the 3D-NPS(fx, fy=0, fz=0), and the NPS(2D-NPS(fx, fy=0)) was different from the 3D-NPS(fx, fy=0, fz=0). To verify them, we compared the NPSs calculated from actual 3D noise data that were obtained using a cylindrical water phantom. RESULTS: The 3D-NPS(fx, fy=0, fz=0) matched the NPSz-projection(fx, fy=0) and was different from the 2D-NPS(fx, fy=0). CONCLUSION: Based on the central slice theorem, we clarified the relationship among NPSs measured by various approaches in NPS analysis; it is important to understand this and then select an appropriate noise data handling and NPS measurement method.

Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution.

Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5'-triphosphate (GTP)-bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the "+" end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.

A cryo-TSEM with temperature cycling capability allows deep sublimation of ice to uncover fine structures in thick cells.

The scanning electron microscope (SEM) has been reassembled into a new type of cryo-electron microscope (cryo-TSEM) by installing a new cryo-transfer holder and anti-contamination trap, which allowed simultaneous acquisition of both transmission images (STEM images) and surface images (SEM images) in the frozen state. The ultimate temperatures of the holder and the trap reached - 190 °C and - 210 °C, respectively, by applying a liquid nitrogen slush. The STEM images at 30 kV were comparable to, or superior to, the images acquired with conventional transmission electron microscope (100 kV TEM) in contrast and sharpness. The unroofing method was used to observe membrane cytoskeletons instead of the frozen section and the FIB methods. Deep sublimation of ice surrounding unroofed cells by regulating temperature enabled to emerge intracellular fine structures in thick frozen cells. Hence, fine structures in the vicinity of the cell membrane such as the cytoskeleton, polyribosome chains and endoplasmic reticulum (ER) became visible. The ER was distributed as a wide, flat structure beneath the cell membrane, forming a large spatial network with tubular ER.

Method for determining slice sensitivity profile of iterative reconstruction CT images using low-contrast sphere phantom.

A novel method for measuring the slice sensitivity profile (SSP) of computed tomography (CT) images reconstructed using an iterative reconstruction (IR) algorithm is proposed herein. A phantom that included a low-contrast spherical object was scanned and consecutive cross-sectional images were reconstructed. The mean CT values in a region including the sphere were measured for all images and plotted as a function of slice position along the longitudinal [Formula: see text] direction to yield a mean CT value profile [Formula: see text]. Next, we numerically generated an object function corresponding to the sphere and obtained the mean CT value profile [Formula: see text]. Subsequently, the SSP was modeled as a product of the Gaussian and cosine functions. We convolved [Formula: see text] with the modeled SSP to obtain [Formula: see text]. The difference between [Formula: see text] and [Formula: see text] was evaluated using the root mean square error (RMSE), which was minimized via optimization of the SSP model parameters. To validate the methodology, we first used filtered back projection (FBP) images to compare the SSPs determined using the proposed and standard coin methods. Subsequently, the proposed method was applied to measure the SSPs of four types of IR algorithms in two scanners. The SSPs of the FBP images determined using the proposed and coin methods showed good agreement. Additionally, in the SSP measurements using the proposed method, [Formula: see text] agreed well with [Formula: see text] for every IR algorithm. The RMSEs for all measurements were less than 0.7 HU, indicating the accuracy of the SSPs. Thus, the proposed method is effective for obtaining valid SSPs.

[Central Slice Theorem-based Relationship between 1D-NPS Obtained by the Slit Method and 2D-NPS for CT Images].

PURPOSE: The method using a numerical slit (slit method) is used commonly to obtain the one-dimensional (1D) noise power spectrum (NPS) in computed tomography. However, the relationship between the 1D-NPS obtained by the slit method and the original two-dimensional (2D) NPS derived by the 2D Fourier transformation has not been elucidated clearly. The purpose of this study was to clarify their relationship based on the well-known central slice theorem (projection slice theorem) and validate it using computer simulation analysis. METHODS: With the application of the central slice theorem, we described that the 1D-NPS obtained by the slit method was equal to the central slice (profile) in the 2D-NPS when we set the slit length to the maximum (i.e. the matrix size of the noise image). To verify this, we generated computer-simulated noise images with the known 2D-NPS (true 2D-NPS). From those images, we obtained the 1D-NPS that was obtained by the slit method and compared it with the central slice in the true 2D-NPS. RESULTS: When we set the slit length to the maximum, the 1D-NPS obtained by the slit method showed good agreement with the central slice in the true 2D-NPS. CONCLUSION: We clarified the relationship between the 1D-NPS obtained by the slit method and the 2D-NPS using a theoretical approach and the computer simulation. We had to maximize the slit length to achieve the accurate measurement of the 1D-NPS using the slit method.

Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization.

Bacterial microcompartments are proteinaceous shells that encase specialized metabolic processes in bacteria. Recent advances in simplification of these intricate shells have encouraged bioengineering efforts. Here, we construct minimal shells derived from the Halothiobacillus neapolitanus α-carboxysome, which we term Cso-shell. Using cryogenic electron microscopy, the atomic-level structures of two shell forms were obtained, reinforcing notions of evolutionarily conserved features in bacterial microcompartment shell architecture. Encapsulation peptide sequences that facilitate loading of heterologous protein cargo within the shells were identified. We further provide a first demonstration in utilizing minimal bacterial microcompartment-derived shells for hosting heterologous enzymes. Cso-shells were found to stabilize enzymatic activities against heat shock, presence of methanol co-solvent, consecutive freeze-thawing, and alkaline environments. This study yields insights into α-carboxysome assembly and advances the utility of synthetic bacterial microcompartments as nanoreactors capable of stabilizing enzymes with varied properties and reaction chemistries.

[A Simple Method for Computationally Generating Metal Artifacts in CT Images for Treatment Planning: A Pilot Phantom Study].

PURPOSE: In treatment planning for radiation therapy, the use of computed tomography (CT) images including metal artifacts causes a reduction in the dose calculation accuracy. In clinical practice, the artifacts are manually contoured and assigned an appropriate fixed CT number. To validate the procedure, images taken before and after metal insertion into a patient are required, which may be impractical. We propose a simple method for computationally generating metal artifacts in clinical images. METHODS: In the proposed method, a clinical image free of metal artifacts is used. To simulate metal inside a patient, CT numbers of a region in the image are replaced with a fixed extremely high value. A sinogram is created by the forward projection of the image. Data values of the sinogram in the metal region are converted into smaller values. From the sinogram, an image including artifacts is reconstructed with the filtered back projection. RESULTS: The simulated artifacts consisted of dark and bright bands and were observed to be similar to the actual metal artifacts. CT numbers in multiple small regions of interest in the image obtained by the proposed method showed a good agreement with those in the actual image. CONCLUSION: The proposed method was demonstrated to generate the metal artifacts additionally on the clinical images. The method would be potentially applicable to a validation study for the clinical procedure of manually contouring and assigning CT numbers to metal artifacts.

Structural Insights into the Regulation of Actin Capping Protein by Twinfilin C-terminal Tail.

Twinfilin is a conserved actin regulator that interacts with actin capping protein (CP) via C terminus residues (TWtail) that exhibits sequence similarity with the CP interaction (CPI) motif of CARMIL. Here we report the crystal structure of TWtail in complex with CP. Our structure showed that although TWtail and CARMIL CPI bind CP to an overlapping surface via their middle regions, they exhibit different CP-binding modes at both termini. Consequently, TWtail and CARMIL CPI restrict the CP in distinct conformations of open and closed forms, respectively. Interestingly, V-1, which targets CP away from the TWtail binding site, also favors the open-form CP. Consistently, TWtail forms a stable ternary complex with CP and V-1, a striking contrast to CARMIL CPI, which rapidly dissociates V-1 from CP. Our results demonstrate that TWtail is a unique CP-binding motif that regulates CP in a manner distinct from CARMIL CPI.

Crystal structure of human V-1 in the apo form.

V-1, also known as myotrophin, is a 13 kDa ankyrin-repeat protein that binds and inhibits the heterodimeric actin capping protein (CP), which is a key regulator of cytoskeletal actin dynamics. The crystal structure of V-1 in complex with CP revealed that V-1 recognizes CP via residues spanning several ankyrin repeats. Here, the crystal structure of human V-1 is reported in the absence of the specific ligand at 2.3 Šresolution. In the asymmetric unit, the crystal contains two V-1 monomers that exhibit nearly identical structures (Cα r.m.s.d. of 0.47 Å). The overall structures of the two apo V-1 chains are also highly similar to that of CP-bound V-1 (Cα r.m.s.d.s of <0.50 Å), indicating that CP does not induce a large conformational change in V-1. Detailed structural comparisons using the computational program All Atom Motion Tree revealed that CP binding can be accomplished by minor side-chain rearrangements of several residues. These findings are consistent with the known biological role of V-1, in which it globally inhibits CP in the cytoplasm.