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Many bacteria belonging to the phylum Bacteroidetes move on solid surfaces, which is called gliding motility. In our previous study with the Bacteroidetes gliding bacterium Flavobacterium johnsoniae, we proposed a helical loop track model, where adhesive SprB filaments are propelled along a left-handed closed helical loop on the cell surface. Attachment of SprB to the substratum results in cell movement. In this study, we observed the gliding cell rotating counterclockwise about its axis when viewed from the rear to the advancing direction of the cell, which was consistent with the helical loop track model. Total internal reflection fluorescence microscopy of SprB on a gliding cell revealed that one labeled SprB focus sometimes overtook and passed another SprB focus that was moving in the same direction, suggesting the presence of multiple lanes in the helical loop track. Several electron microscopic analyses revealed the presence of a multi-rail structure underneath the outer membrane, which was associated with SprB filaments and contained GldJ protein. A similar structure was observed in the distantly related marine gliding Bacteroidetes Saprospira grandis. These results provide new insights into the mechanism of Bacteroidetes gliding motility, in which the SprB filaments are propelled along the multi-rails underneath the outer membrane.

Skeletal myosin S1 consists of two functional segments, a catalytic-domain and a lever-arm. Since the crystal structure of ADP/Vi-bound S1 exhibits a strong intramolecular flexure between two segments, inter-conversion between bent and extended forms; i.e. "tilting of the lever-arm" has been accepted as the established molecular mechanism of skeletal muscle contraction. We utilized quick-freeze deep-etch replica electron microscopy to directly visualize the structure of in vitro actin-sliding myosin, and found the existence of a novel oppositely-bent configuration, instead of the expected ADP/Vi-bound form. We also noticed that SH1-SH2 cross-linked myosin gives an aberrant appearance similar to the above structure. Since SH1-SH2-cross-linked myosin is a well-studied analogue of the transient intermediate of the actomyosin cross-bridge cycle, we devised a new image-processing procedure to define the relative view-angles between the catalytic-domain and the lever-arm from those averaged images, and built a 3-D model of the new conformer. The lever-arm in that model was bent oppositely to the ADP/Vi-bound form, in accordance with observed actin-sliding cross-bridge structure. Introducing this conformer as the crucial intermediate that transiently appears during sliding, we propose a revised scheme of the cross-bridge cycle. In the scenario, the novel conformer keeps actin-binding in two different modes until it forms a primed configuration. The final extension of the lever-arm back to the original rigor-state constitutes the "power-stroke". Various images observed during sliding could be easily interpreted by the new conformer. Even the enigmatic behavior of the cross-bridges reported as "loose chemo-mechanical coupling" might be adequately explained under some assumptions.

3-D Structural information is essential to elucidate the molecular mechanisms of various biological machineries. Quick-Freeze Deep-Etch-Replica Electron Microscopy is a unique technique to give very high-contrast surface profiles of extra- and intra-cellular apparatuses that bear numerous cellular functions. Though the global architecture of those machineries is primarily required to understand their functional features, it is difficult or even impossible to depict side- or highly-oblique views of the same targets by usual goniometry, inasmuch as the objects (e.g. motile microorganisms) are placed on conventional flat substrates. We introduced silica-beads as an alternative substrate to solve such crucial issue. Elongated Flavobacterium and globular Mycoplasmas cells glided regularly along the bead’s surface, similarly to those on a flat substrate. Quick-freeze replicas of those cells attached to the beads showed various views; side-, oblique- and frontal-views, enabling us to study not only global but potentially more detailed morphology of complicated architecture. Adhesion of the targets to the convex surface could give surplus merits to visualizing intriguing molecular assemblies within the cells, which is relevant to a variety of motility machinery of microorganisms.

Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed. IMPORTANCE Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae . This tiny, 2-μm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma “gliding motility” is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism.

In this work, we examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica electron microscopy (EM) by using a new method of image processing/analysis based on mathematical morphology. In order to quantify the degree of structural changes, two characteristic patterns were extracted from the EM images. One is the winding pattern of the filament shape (WP) reflecting flexibility of the filament, and the other is the surface pattern of the filament (SP) reflecting intra-molecular domain-mobility of actin monomers constituting the filament. EM images were processed by morphological filtering followed by box-counting to calculate the fractal dimensions for WP (DWP) and SP (DSP). The result indicates that DWP was larger than DSP irrespective of the state of the filament (myosin-free or bound) and that both parameters for myosin-bound filaments were significantly larger than those for myosin-free filaments. Overall, this work provides the first quantitative insight into how conformational disorder of actin monomers is correlated with the myosin-induced increase in flexibility of actin filaments along their length as suggested by earlier studies with different techniques. Our method is yet to be improved in details, but promising as a powerful tool for studying the structural change of protein molecules and their assemblies, which can potentially be applied to a wide range of biological and biomedical images.

Various proteins are known to exhibit one-dimensional Brownian motion along charged rodlike polymers, such as microtubules (MTs), actin, and DNA. The electrostatic interaction between the proteins and the rodlike polymers appears to be crucial for one-dimensional Brownian motion, although the underlying mechanism has not been fully clarified. We examined the interactions of positively-charged nanoparticles composed of polyacrylamide gels with MTs. These hydrophilic nanoparticles bound to MTs and displayed one-dimensional Brownian motion in a charge-dependent manner, which indicates that nonspecific electrostatic interaction is sufficient for one-dimensional Brownian motion. The diffusion coefficient decreased exponentially with an increasing particle charge (with the exponent being 0.10 kBT per charge), whereas the duration of the interaction increased exponentially (exponent of 0.22 kBT per charge). These results can be explained semiquantitatively if one assumes that a particle repeats a cycle of binding to and movement along an MT until it finally dissociates from the MT. During the movement, a particle is still electrostatically constrained in the potential valley surrounding the MT. This entire process can be described by a three-state model analogous to the Michaelis-Menten scheme, in which the two parameters of the equilibrium constant between binding and movement, and the rate of dissociation from the MT, are derived as a function of the particle charge density. This study highlights the possibility that the weak binding interactions between proteins and rodlike polymers, e.g., MTs, are mediated by a similar, nonspecific charge-dependent mechanism.

In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca(2+)-dependent reversible binding of K560-CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560-CaM in the absence of Ca(2+), while both dimeric and tetrameric configurations in the presence of Ca(2+). Further, Ca(2+)-dependent change in the configuration of K560-CaM was monitored by size-exclusion-HPLC and analytical ultracentrifugation. Finally, by total internal reflection fluorescence microscopy, we successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca(2+).

アニオン交換多孔性中空糸膜に血漿中のタンパク質を吸着させた後, カルシウムイオンを含む液を使って膜からgelsolinを特異的に溶出させた。エポキシ基を有するグラフト鎖を多孔性中空糸膜に付与した後, ジエチルアミン, 引き続いてエタノールアミンを反応させて, 2種類のアニオン交換基として, ジエチルアミノ (DEA) 基と2-ヒドロキシエチルアミノ (HEA) 基をグラフト鎖に導入した。グラフト鎖中のDEA基のモル分率を0.53まで変化させ, 膜の透過流束とgelsolin回収率を測定した結果, HEA基のみを有するグラフト鎖を搭載した多孔性中空糸膜が最適であることがわかった。また, gelsolin精製について, アニオン交換多孔性中空糸膜とアニオン交換ビーズカラムとで性能を比較した。多孔性中空糸膜を使うと, タンパク質溶液の空間速度を高くしても, gelsolin回収率が一定である点で有利であることを示した。

Some pathogenic bacteria actually invade the cytoplasm of their target host cells. Invasive bacteria acquire the propulsive force to move by recruiting actin and inducing its polymerization. Here we show thatShigellamovement within the cytoplasm was severely hindered by microtubules and that the bacteria destroyed surrounding microtubules by secreting VirA by means of the type III secretion system. Degradation of microtubules by VirA was dependent on its α-tubulin–specific cysteine protease–like activity.virAmutants did not move within the host cytoplasm and failed to move into adjacent cells.

Class VI myosin is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. Recently, however, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting a doubt on its processivity. By using single molecule techniques, we show that green-fluorescent-protein-tagged single-headed, wild-type myosin-VI does not move processively. However, when coupled to 200-nm polystyrene beads (comparable to intracellular vesicles in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40-nm) steps. The characteristics of this monomer-driven movement were different to that of artificial dimer-driven movement: Compared to the artificial dimer, the monomer-bead complex had a reduced stall force (1pN compared to 2pN), an average run length 2.5-fold shorter (91nm compared to 220nm) and load-dependent step size. Furthermore, we found that a monomer-bead complex moved more processively in a high viscous solution (40-fold higher than water) similar to cellular environment. Because the diffusion constant of the bead is 60-fold lower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing its rebinding following detachment and supporting processive movement of the bead-monomer complexes. Although a single-headed myosin-VI was able to move processively with a large cargo, the travel distance was rather short. Multiple molecules may be involved in the cargo transport for a long travel distance in cells.

Type III secretion machinery (TTSM), composed of a needle, a basal body, and a C-ring compartment, delivers a subset of effectors into host cells. Here, we show that Shigella Spa33 is an essential component of the C-ring compartment involved in mediating the transit of various TTSM-associated translocated proteins. Electron microscopic analysis and pull-down assay revealed Spa33 to be localized beneath the TTSM via interaction with MxiG and MxiJ (basal body components). Spa33 is also capable of interacting with Spa47 (TTSM ATPase), MxiK, MxiN (required for the transit of MxiH, the needle component), Spa32 (required for determining needle length), and several effectors. Genetic and functional analyses of the Spa33 C-terminal region, which is highly conserved in the SpaO-YscQ-HrcQ(B)-FliN family, indicate that some of the conserved residues are crucial for needle formation via interactions with MxiN. Thus, Spa33 plays a central role as the C-ring component in recruiting/exporting TTSM-associated proteins.

Acto-S1 chimera proteins CP24 and CP18 carry the entire actin sequence, inserted in loop 2 of the motor domain of Dictyostelium myosin II, and have MgATPase activity close to that of natural Dictyostelium actomyosin [M.S.P. Siddique, T. Miyazaki, E. Katayama, T.Q.P. Uyeda, M. Suzuki, Evidence against essential roles of subdomain 1 of actin in actomyosin sliding movements, Biochem. Biophys. Res. Commun. 332 (2005) 474–481]. Here, we examined and detected cooperative structural change of actin filaments accompanying interaction with myosin motor domain in the presence of ATP using copolymer filaments consisting of pyrene-labeled skeletal actin (SA) and either CP24 or CP18. Upon addition of ATP, the fluorescence intensity increased over the range from 380 to 480 nm using 365- nm excitation. The relative increases of fluorescence intensity at 390 nm were 14%, 46%, and 77% for the copolymer filaments with the CP24 to actin molar ratios of 0.0625, 0.143, and 0.333, respectively, and demonstrated a sigmoid behavior. Stoichiometric analysis indicates that each CP24 molecule appears to affect four actin molecules, on average, in SA-CP24 copolymers, and each CP18 molecule appears to affect three actin molecules in SA-CP18 copolymers.

Gelsolin was purified from bovine plasma using an anion-exchange porous hollow-fiber membrane. The anion-change porous hollow-fiber membrane was prepared by radiation-induced graft polymerization of an epoxy-group-containing monomer, glycidyl methacrylate, and subsequent chemical modifications. Some of the epoxy groups of the polymer chain grafted onto the pore surface were converted into diethylamino groups, and the remaining epoxy groups were converted into 2-hydroxyethylamino groups. First, a gelsolin-containing dialyzed protein solution, prepared by pretreatments of ammonium sulfate precipitation and dialysis of plasma, was forced to permeate through the pores of an anion-exchange porous hollow-fiber membrane. Various proteins including gelsolin were adsorbed onto the anion-exchange polymer brush at a high rate with negligible diffusional mass-transfer resistance. Second, adsorbed gelsolin was specifically eluted by permeating 2 mM calcium chloride. The amount of recovered gelsolin was 0.1 mg per 1 mL of plasma. Third, the remaining adsorbed proteins were quantitatively eluted with 1 M sodium chloride, leading to a constant amount of recovered gelsolin during four cycles of purification. The total time required for gelsolin purification from 30 mL of bovine plasma was 11 h, during which the time for selective adsorption of various proteins and affinity elution of gelsolin using the anion-exchange porous hollow-fiber membrane was 20 min.

Myosin forms stable ternary complexes with ADP and phosphate analogues of fluoro-metals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin-ADP-fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM).ADP.fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM. ADP-BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM-ADP-fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.

A characteristic feature of the astrocytic processes is to assume the form of shin sheets or lamellate coverings of other brain constituents. We analyzed the extensive and finely divided processes of the protoplasmic astrocyte in the molecular layer of the rat dentate gyrus by means of computer electron tomography and stereo-photogrammetry using tilted high voltage electron microscope images of thick Golgi preparations. The surface area and volume of the astrocytic processes were measured and the surface/volume ratios were estimated. The surface/volume ratios of astrocytic processes in the neuropile ranged from 18.9 to 33.0 per microm, and the mean value was 26.2+/-5.0 per microm. The values were roughly comparable to those previously reported for the microdomain of Bergmann glia cell terminal processes in the rat cerebellum, which were estimated from reconstructions using thin serial section electron microscope images. The large surface to volume ratio of the astrocytic processes in the neuropile resulted from the lamellar nature of the processes interposed between other cellular elements, and may reflect the functional activities of the astrocyte. The results suggest the usefulness of the electron tomography and stereo-photogrammetry for three-dimensional morphometrical analysis of the astrocytic processes, although both techniques can be expected to be refined further in order to provide more precise measurements of these complicated processes.

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001)J. Biol. Chem. 276, 27670–27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca2+-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the β4 strands of the C2 structures (so-called “C2 effector domain”) is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is “a large linear structure,” not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.

The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri , the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 μm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ , encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.

High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries. DOI: http://dx.doi.org/10.7554/eLife.04806.001, eLife digest Actin is one of the most abundant proteins found inside all eukaryotic cells including plant, animal, and fungal cells. This protein is involved in a wide range of biological processes that are essential for an organism's survival. Actin proteins form long filaments that help cells to maintain their shape and also provide the force required for cells to divide and/or move. Actin filaments are helical in shape and are made up of many actin subunits joined together. Actin filaments are changeable structures that continuously grow and shrink as new actin subunits are added to or removed from the ends of the filaments. One end of an actin filament grows faster than the other; the fast-growing end is known as the barbed-end, while the slow-growing end is referred to as the pointed-end. Over 100 other proteins are known to bind to and work with actin to regulate its roles in cells and how it forms into filaments. Cofilin is one such protein that binds to and forms clusters on actin filaments and it can also sever actin filaments. Severing an actin filament can encourage the filament to disassemble, or it can help produce new barbed ends that can then grow into new filaments. Previous work had suggested that cofilin severs actin filaments at the junction between regions on the filament that are coated with cofilin and those that are not. It was also known that cofilin binding to a filament causes the filament to change shape, and that the shape change is propagated to neighboring sections of the filaments not coated with cofilin. However, the details of where cofilin binds and how changes in shape are propagated along an actin filament were not known. Furthermore, the findings of these previous studies were largely based on examining still images of actin filaments, which are unlike the constantly changing filaments of living cells. Ngo, Kodera et al. have now analyzed what happens when cofilin binds to and forms clusters along actin filaments using a recently developed imaging technique called high-speed atomic force microscopy. This technique can be used to directly visualize individual proteins in action. Consistent with previous findings, Ngo, Kodera et al. observed that filaments coated with cofilin are thicker than those filaments without cofilin; and that cofilin binding also substantially reduces the helical twist of the filament. Ngo, Kodera et al. also found that these changes in shape are propagated along the filament but in only one direction—towards the pointed-end. Moreover, cofilin clusters also only grew towards the pointed-end of the actin filament—and the filaments were often severed near, but not exactly at, the junctions between cofilin-coated and uncoated regions. Such one-directional changes in shape of the actin filaments presumably help to regulate how other actin binding proteins can interact with the filament and consequently regulate the roles of the filaments themselves. DOI: http://dx.doi.org/10.7554/eLife.04806.002

The BRCT regions are two repeating structures in BRCA1 at the carboxyl-terminus and are ubiquitous in some proteins involved in cell cycle checkpoint and in DNA repair. Here, using electron microscopy, we show direct evidence that the BRCT regions of BRCA1 bound double-strand breaks of DNA. The BRCT regions could multimerize thus forming large protein particles. Smeared patterns of DNA fragments were consistently shown in the gel retardation assay. A single BRCT was sufficient for DNA binding. The smeared patterns were also observed in BRCTs of TopBP1, suggesting that multimerization may be an important feature of BRCTs. The recombinant second BRCT of XRCC1 (X-ray repair cross-complementing group 1), whose folding was determined by X-ray crystallography, also showed similar DNA end binding images. It is possible that some BRCTs are fundamental structures that detect DNA damages.

We investigated the supramolecular structure of the Shigella type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 ± 3.3 and 31.6 ± 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47 , encoding a putative F 1 ‐type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle‐less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.

Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both in vitro and in vivo . The Reelin–Reelin interaction clearly is inhibited by the function-blocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment.

The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. After screening for promoters exhibiting bfpTVW-dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non-virulent E. coli laboratory strains, DH5alpha and HB101. Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E. coli K12. This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW-activated promoter was localized upstream of ORF1. An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription. An ORF1-His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone-like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene). The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment.

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (alpha-1,6-glucan) into a water-insoluble form by making numerous alpha-1,3-glucan branches along the dextran chains with sucrose as the glucosyl donor. The structures and catalytic properties were compared for two GTF-I fragments, GTF-I' (138 kDa) and GS (110 kDa). Both lack the N-terminal 84 residues of GTF-I. While GTF-I' still contains four of the six C-terminal repeats characteristic of streptococcal glucosyltransferases, GS lacks all of them. Electron microscopy of negatively stained samples indicated a double-domain structure for GTF-I', consisting of a spherical head with a smaller spherical tail, which was occasionally seen as a long extension. GS was seen just as the head portion of GTF-I'. In the absence of dextran, both fragments simply hydrolyzed sucrose with similar K(m) and k(cat) values at low concentrations (5 mM). At higher sucrose concentrations (10 mM), however, GTF-I' exhibited glucosyl transfer activity to form insoluble alpha-1, 3-glucans. So did GS, but less efficiently. Dextran increased the rate and efficiency of the glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF-I' by mild trypsin treatment, this dextran-stimulated transfer was completely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are organized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form alpha-1,3-glucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as well as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.

Eukaryotic flagella beat rhythmically. Dynein is a protein that powers flagellar motion, and oscillation may be inherent to this protein. Here we determine whether oscillation is a property of dynein arms themselves or whether oscillation requires an intact axoneme, which is the central core of the flagellum and consists of a regular array of microtubules. Using optical trapping nanometry, we measured the force generated by a few dynein arms on an isolated doublet microtubule. When the dynein arms on the doublet microtubule contact a singlet microtubule and are activated by photolysis of caged ATP8, they generate a peak force of approximately 6pN and move the singlet microtubule over the doublet microtubule in a processive manner. The force and displacement oscillate with a peak-to-peak force and amplitude of approximately 2 pN and approximately 30 nm, respectively. The geometry of the interaction indicates that very few (possibly one) dynein arms are needed to generate the oscillation. The maximum frequency of the oscillation at 0.75 mM ATP is approximately 70 Hz; this frequency decreases as the ATP concentration decreases. A similar oscillatory force is also generated by inner dynein arms alone on doublet microtubules that are depleted of outer dynein arms. The oscillation of the dynein arm may be a basic mechanism underlying flagellar beating.

We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.

The precise geometry of the flagellar basal structure anchored in the cytoplasmic membrane was determined by digital stereo-photogrammetry of the images captured by quick-freeze deep-etch replica electron microscopy. In order to examine the structure on the periplasmic side of the membrane, we analyzed the MS ring complexes ofSalmonella typhimuriumoverproduced in the cytoplasmic membrane ofEscherichia coli. The rod, the S ring, and the shoulder of the M ring were exposed to the periplasm. On the cytoplasmic side of the membrane, small bumps corresponding to the cytoplasmic rod were discernible. We also examined the intact inner surface of the cells of polyhook mutant which was prepared by a new protocol and found the bell-shaped structure extending from the membrane towards the cytoplasm. It was identified as the C ring, since it was located at the base of the polyhook. Various dimensions of the MS ring complex and the C ring projecting from the membrane were determined by digital stereo-photo grammetry, and a three-dimensional model of the total basal structure is presented.

In the present paper, we described our attempt to characterize the rough three-dimensional features of the structural analogue of the key intermediate of myosin's cross-bridge cycle. Using quick-freeze deep-etch replica electron microscopy, we observed that actin-attached myosin during in vitro sliding was bent superficially as postulated by the conventional hypothesis, but in the opposite direction of the putative pre-power-stroke configuration, as for ADP·Vi (inorganic vanadate)-bound myosin. We searched for the conformational species with a similar appearance and found that SH1–SH2 (thiols 1 and 2)-cross-linked myosin is a good candidate. To characterize such small asymmetric structures, we employed a new pattern-recognition procedure that accommodates the metal-replicated samples. In this method, the best-matched views of the target microscopic images were selected from a comprehensive set of images simulated from known atomic co-ordinates of relevant proteins. Together with effective morphological filtering, we could define the conformational species and the view angles of the catalytic domain and the lever arm cropped from averaged images of disulfide-cross-linked myosin. Whereas the catalytic domain of the new conformer closely resembled the pPDM (N,N′-p-phenylenedimaleimide)-treated, but SH2 Lys705-cross-linked, structure (PDB code 1L2O), a minor product of the same cross-linking reaction, the lever arm projected differently. Using separately determined view angles of the catalytic domain and the lever arm, we built a model of disulfide-cross-linked myosin. Further combination with the ‘displacement-mapping’ procedure enabled us to reconstruct the global three-dimensional envelope of the unusual structure whose lever arm orientation is compatible with our reports on the actin-sliding cross-bridge structure. Assuming this conformer as the structural analogue of the transient intermediate during actin sliding, the power stroke of the lever arm might accompany the reversal of the disorganized SH1 helix.

The structure of smooth muscle thin filament was examined by various electron microscopy techniques, with special attention to the mode of caldesmon binding. Chemical cross-linking was positively used to avoid the dissociation of accessory proteins upon dilution. Caldesmon in reconstituted thin filament was observed as fine filamentous projections from thin filament. Native thin filament isolated from smooth muscle showed similarly numerous fine whisker-like projections by all the techniques employed here. Antibody against the amino-terminus of caldesmon labeled the end of such projections indicating the possibility that the amino-terminal myosin binding moiety might stick out from the shaft of the thin filament. Such whiskers are often projected out as a cluster to the same side of native thin filament. Further, we could visualize the assembly of dephosphorylated heavy meromyosin (HMM) with native or reconstituted thin filament forming "nonproductive" complex in the presence of ATP. The association of HMM to the shaft of thin filament was through subfragment-2 moiety, in accordance with biochemical studies. Some HMM particles bound closer to the thin filament shaft, possibly suggesting the presence of the second myosin-binding site on caldesmon. Occasionally two kinds of HMM association as such coexisted at a single site on this filament in tandem. Thus, we constructed a structural model of thin filament. The proposed molecular arrangement is not only compatible with all the biochemical results but also provides additional support for our recent findings (E. Katayoma, G. C. Scott-Woo, and M. Ikebe (1995) J. Biol. Chem. 270, 3919-3925) regarding the capability of caldesmon to induce dephosphorylated myosin filament, which explains the existence of thick filaments in relaxed smooth muscle cells.

The large plasmid-encoded outer membrane protein VirG (IcsA) of Shigella flexneri is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that VirG is located at one pole on the surface of the bacterium and secreted into the culture supernatant and that in host cells it is localized along the length of the F-actin tail. The roles of these VirG phenotypes in bacterial spreading still remain to be elucidated. In this study, we examined the surface-exposed portion of the VirG protein by limited trypsin digestion of S. flexneri YSH6000 and determined the sites for VirG processing during secretion into the culture supernatant. Our results indicated that the 85-kDa amino-terminal portion of VirG is located on the external side of the outer membrane, while the 37-kDa carboxy-terminal portion is embedded in it. The VirG cleavage required for release of the 85-kDa protein into the culture supernatant occurred at the Arg-Arg bond at positions 758 to 759. VirG-specific cleavage was observed in Shigella species and enteroinvasive Escherichia coli, which requires an as yet unidentified protease activity governed by the virB gene on the large plasmid. To investigate whether the VirG-specific cleavage occurring in extracellular and intracellular bacteria is essential for VirG function in bacterial spreading, the Arg-Arg cleavage site was modified to an Arg-Asp or Asp-Asp bond. The virG mutants thus constructed were capable of unipolar deposition of VirG on the bacterial surface but were unable to cleave VirG under in vitro or in vivo conditions. However, these mutants were still capable of eliciting aggregation of F-actin at one pole, spreading into adjacent cells, and giving rise to a positive Sereny test. Therefore, the ability to cleave and secrete VirG in Shigella species is not a prerequisite for intracellular spreading.

Images of shadowed F-actin filaments on mica surfaces obtained using a quick-freeze, freeze-fracture, deep-etch technique were subjected to conventional 3-D helical reconstruction methods. Although the shadowing must vary systematically from subunit to subunit, the computed transforms of isolated filaments were characteristic of the helical actin transform. Helical reconstruction was therefore judged to be valid. The theoretical basis for such reconstruction is outlined. The reconstructions showed an average thin (about 3 nm) layer of shadow on the filament surface and both the outer and the inner surfaces of the shadow layer could be visualized. By comparison with the F-actin structure postulated by Holmes et al. (1990) on the basis of the known structure of the actin monomer, it is shown that, at the resolution considered, the inner surface of the shadow provides a reasonably faithful outline of the molecular surface. This, in turn, confirms that the original 3-D structure of the protein molecules has been well preserved throughout the whole preparation procedure up to the final replica. The "shadowed" filaments can thus be correlated axially and azimuthally with known actin structures and, in principle, features such as myosin head location on decorated filaments can be determined. The result emphasizes the amount of detail present in good quality images of shadowed particles and, in this case, shows that detailed evaluation of molecules labeling actin can be made.

Aqueous uranyl acetate has been extensively used as a superb staining reagent for transmission electron microscopy of biological materials. However, recent regulation of nuclear fuel material severely restricts its use even for purely scientific purposes. Since uranyl salts are hazardous due to biological toxicity and remaining radioactivity, development of safe and non-radioactive substitutes is greatly anticipated. We examined two lanthanide salts, samarium triacetate and gadolinium triacetate, and found that 1-10% solution of these reagents was safe but still possess excellent capability for staining thin sections of plastic-embedded materials of animal and plant origin. Although post-fixation with osmium tetroxide was essential for high-contrast staining, post-staining with lead citrate could be eliminated if a slow-scan CCD camera is available for observation. These lanthanide salts can also be utilized as good negative-staining reagents to study supramolecular architecture of biological macromolecules. They were not as effective as a fixative of protein assembly, reflecting the non-hazardous nature of the reagents.

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

We have been investigating conformational changes of myosin crossbridges during in vitro sliding, utilizing quick-freeze-replication and a novel image analysis to quantitatively compare microscopic images with the atomic models of various conformations. We reported that the leverarm moiety of working crossbridges, is mostly kinked to the opposite side of ADP/Vi-bound structure and such global configuration of myosin head resembles that whose SH1 and −2 are chemically crosslinked. Since the crystal structure of such unusual configuration is not determined, we attempted to reconstruct its 3-D structure by a special version of Single-Particle-Analysis, devised to adapt small-sized particle, utilizing very contrasty feature of metal-replicated images. With a new procedure to manage very few view-angles, we finally obtained the 3-D envelope of the myosin head with oppositely kinked leverarm, analogous to the intermediate structure under sliding conditions. We cut the original atomic model of pPDM-treated-ADP-structure (1L20) into motor-domain and the leverarm, and relocated each module at best-matching position and the orientation, to generate a tentative model that best-fits to reconstructed envelope. We then examined whether all the images of actin-sliding crossbriges can be uniquely explained by that standard SH-crosslinked structure model. The images of actin-attached crossbridges were classified by “2-D appearance” and each class-average was compared with 2-D projections of the standard structure as above. By analyzing the orientation of motor-domain and leverarm separately, we found that there could be several sub-populations, some matched to but some deviated from the standard structure. It is known that two reactive thiols can be crosslinked by a variety of bifunctional reagents of different span lengths, implying the distance between them might be flexible. We assume that the extension of new oppositely-kinked configuration would comprise the power-stroke and those sub-populations might correspond to several steps during that structural change.

In the present study, we have demonstrated that the novel molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. We successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca2+ by the total internal reflection fluorescence microscopy. Reversible Ca2+dependent cargo-loading system was achieved by changing the Ca2+ concentration in the flow cell. K560-CaM was adsorbed onto the fluorescently unlabeled microtubule adhered on the glass surface in flow cell using non-hydrolyzable ATP analogue, AMP-PNP which stabilize the microtubule binding state of kinesin. Subsequently, Qdot-M13 was added in the presence of Ca2+ to be loaded on K560-CaM adsorbed on the microtubule. The fluorescence of Qdot-M13 loaded onto K560-CaM along a microtubule was observed after washing excess unbound Qdot-M13. When the Ca2+ solution in the flow cell was replaced by the Ca2+ free solution, Qdot-M13 was unloaded. Even after the several times alternate exchange of the solution in the flow cell with Ca2+ and EGTA solutions, the K560-CaM adsorbed onto microtubule by AMP-PNP showed stable Ca2+ dependent cargo loading. When excess ATP was added into the flow cell, K560-CaM-Qdot started to move along the microtubule. Interestingly, 145 seconds later, K560-CaM-Qdot accumulated at plus end of microtubule and showed fluorescent clumps as marker for plus end of microtubule.

To investigate the relationship between the mechanical and biochemical properties of cardiac myosin, the sliding velocity of isolated cardiac myosin obtained from both euthyroid and hyperthyroid rabbits on actin cables was measured with an in vitro motility assay system. Ten rabbits (T) were treated with L-thyroxine to induce hyperthyroidism, and eight nontreated animals (N) were used as controls. Myosin was purified from the left ventricles of anesthetized animals. Myosin isozyme content was analyzed by the pyrophosphate gel electrophoresis method, and myosin adenosinetriphosphatase (ATPase) activity was determined on the same sample. Long well-organized actin cables of green algae, Nitellopsis, were used in the in vitro motility assay. Small latex beads were coated with purified cardiac myosin and introduced onto the Nitellopsis actin cables. Active unidirectional movement of the beads on the actin cables was observed under a photomicroscope, and the velocity was measured. The velocity was dependent on ATP concentrations, and the optimal pH for bead movement was approximately 7.0-7.5. The mean velocity was higher in T than in N (0.66 +/- 0.12 vs. 0.32 +/- 0.09 micron/s, P less than 0.01). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also higher in T than in N (0.691 +/- 0.072 vs. 0.335 +/- 0.072 microM Pi.mg-1.min-1, P less than 0.01, and 79 +/- 12 vs. 26 +/- 7%, P less than 0.01, respectively). The velocity of myosin closely correlated with both Ca(+2)-activated myosin ATPase activity (r = 0.87, P less than 0.01) and the percentage of alpha-myosin heavy chain (r = 0.87, P less than 0.01).

Previously biochemical studies have demonstrated that the highly reactive cysteine residues SH1 and SH2 can be crosslinked by variety of bifunctional reagents with different spans (3-14 A) in the presence of nucleotides, suggesting that the region is highly flexible. The SH1-SH2 region is believed to play a key role in the conformational changes that occur in the myosin head during the force generation coupled to ATP hydrolysis. We have previously shown that the HMM, which SH1-SH2 was crosslinked by p-Phenylene-dimaleimide (pPDM) in the presence of ADP, have a novel conformation using quick freeze deep etch electron microscopy (QFDE-EM). We have also demonstrated that conformational change of the myosin motor domain during ATP hydrolysis can be monitored by measuring the FRET using fluorescent ATP analogue NBD-ATP. In the present study, we analyzed the conformation of the myosin crosslinked by pPDM using the FRET between the ATP binding site and the A1 essential light chain (ELC) and compared with the 3D structure models of ATPase intermediates derived from electron microscopic analysis. We prepared the skeletal muscle myosin subfragment-1 (S1), which ELC was labeled by 6-bromoacetyl-2-dimethylaminonaphthalene (BD) at the Cys 177. And fluorescent ADP analogue NBD-ADP was trapped in the ATPase site of S1 labeled by BD. The FRET efficiency was estimated by measuring the change of fluorescence intensity of BD comparing with control BD-S1. FRET efficiency of pPDM-S1-NBD-ADP was apparently different from other nucleotides and nucleotide analogues bound states.

Electron tomography by conventional filtered back-projection is often seriously impaired by anisotropic resolution due to unavoidable limitation in specimen tilt-angles. We propose a new approach to overcome the problem for thin film-like replica-type specimens in which internal density is supposed as homogenous and contiguously distributed, by imposing a reasonable constraint of density-existing region in the reconstruction procedure. The objects were approximated as a distribution of binary voxels and the intensity of the projected images being proportional to the thickness along the projection ray. The new reconstruction algorithm consists of initial determination of approximate constraint region by a topographic analysis by stereo-photogrammetry, followed by iterative computation to find the unique solution of simultaneous equations, so that all the intensity distribution in tilt-series images are included within pre-determined voxel arrangement. During a trial run with a new methodology, we realized its significantly advantageous feature that much less number of projection images than conventional back-projection is required to perform the reconstruction of almost equivalent quality. Here, we show the performance of this novel algorithm by 3-D reconstruction of quick-freeze deep-etch replica specimens without any trace of spurious ghosting caused by missing-wedge problems.