Quick-freeze Deep-etch Electron Research Articles

Visualization of Bacillus subtilis spore structure and germination using quick-freeze deep-etch electron microscopy.

Bacterial spores, known for their complex and resilient structures, have been the focus of visualization using various methodologies. In this study, we applied quick-freeze and replica electron microscopy techniques, allowing observation of Bacillus subtilis spores in high-contrast and three-dimensional detail. This method facilitated visualization of the spore structure with enhanced resolution and provided new insights into the spores and their germination processes. We identified and described five distinct structures: (i) hair-like structures on the spore surface, (ii) spike formation on the surface of lysozyme-treated spores, (iii) the fractured appearance of the spore cortex during germination, (iv) potential connections between small vesicles and the core membrane and (v) the evolving surface structure of nascent vegetative cells during germination.

Architecture of Actinoplanes missouriensis sporangia and zoospores visualized using quick-freeze deep-etch electron microscopy.

The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface.

Visualization of Peptidoglycan Structures of Escherichia coli by Quick-Freeze Deep-Etch Electron Microscopy.

Peptidoglycan (PG) is an essential component of the bacterial cell wall that protects the cell from turgor pressure and maintains its shape. In diderm (gram-negative) bacteria, such as Escherichia coli, the PG layer is flexible with a thickness of a 2-6nm, and its visualization is difficult due to the presence of the outer membrane. The quick-freeze deep-etch replica method has been widely used for the visualization of flexible structures in cell interior, such as cell organelles and membrane components. In this technique, a platinum replica on the surface of a specimen fixed by freezing is observed using a transmission electron microscope. In this chapter, we describe the application of this method for visualizing the E. coli PG layer. We expect that these methods will be useful for the visualization of the PG layer in diverse bacterial species.

Ultrastructure of the foliose lichen Myelochroa leucotyliza and its solo fungal and algal (Trebouxia sp.) partners

Lichens are slow-growing niche-constructing organisms formed via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. We have used quick-freeze deep-etch electron microscopy (QFDEEM), coupled with several additional microscopic techniques, to analyze the Japanese foliose lichen Myelochroa leucotyliza. We have also examined its constituent fungi and algae grown separately (solo) and in co-culture in the laboratory, as well as solo fungi incubated with conditioned algal media (CM) for 9 days. We report our general findings on M. leucotyliza architecture in a Supplement, and focus this report on three findings of particular interest. 1) Plasma membrane differentiations called nubbins are found adjacent to, and at the center of, the cell-wall septa in both lichenized and solo hyphae, suggesting a role in cell polarity determination and/or cell-cell communication within the hyphae. 2) Nubbins also localize to the tips of hyphal sub-branches produced by the lichenized but not the solo fungi. These in turn form tip-to-tip contacts (bridges) with one another, suggesting a role in inter-hyphal communication. 3) CM treatment of solo fungi induces a) the production of secondary metabolites, polysaccharides, and lipids; b) a transformation of ER topology and its association with the plasma membrane; and c) a dramatic change in vacuole organization, indicating that the algal growth medium contains soluble materials that trigger fungal differentiation.

Lichen 2. Constituents

Lichens are slow-growing niche-constructing organisms that form a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM) to document the ultrastructure of the eukaryotic organisms and the extracellular matrix materials that are found in four lichens. Previous studies have shown that each thallus in a complex lichen consists of a central medullary layer containing aligned fungal hyphae. These medullary hyphae send lateral branches to an upper layer, some making contact with resident algae, and usually to a lower layer. As the thallus ages, such “regular” branches transform into acellular versions called “struts” and “honeycombs.” We conclude with a consideration of two materials that are synthesized and secreted by lichenized fungi: abundant secondary products/metabolites that often crystallize, and hydrophobin proteins that self-assemble into films.

Lichen 4. The Algal Layer

A lichen is a slow-growing niche-constructing organism that forms a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM) and light microscopy to probe a hallmark lichen construction wherein clusters of algae and hyphae, inter-connected by wall-to-wall junctions, form stable consortia that we call green modules. These assemble in the pseudo-meristem and then localize to the algal layer of the thallus. In the foliose lobes of Candelaria concolor, the cells in each module are enveloped in a continuous 10-nm-thick film of hydrophobin proteins, which binds to wall and matrix surfaces on its hydrophilic side and faces air or water on its hydrophobic side. We document patterned relationships between modules and associated cords of hyphae destined for the outer layers, and propose ways that these relationships could form the structural foundation for water and air regulation within foliose lobes.

Lichen 1. Solo fungal and algal partners

Lichens are slow-growing niche-constructing organisms that form a thallus via scripted symbiotic/mutualistic relationships between fungi, algae, and bacteria, and that are distributed across nearly all terrestrial ecosystems. Here we use quick-freeze deep-etch electron microscopy (QFDEEM) to probe the ultrastructure of a lichen-forming fungus, Cladonia grayi, and its partner alga, Asterochloris glomerata, grown separately (solo) in the laboratory. The solo fungus resembles its lichenized counterpart in: a) general cellular organization; b) the capacity to form lateral branches; c) the production of extracellular materials; and d) the generation of acellular hyphae that we call struts. It differs in carrying a fibrillar coat on its wall exterior and in often adopting two novel plasma-membrane configurations called pleated and pitted. The solo alga also resembles its lichenized counterpart in general cellular organization, but its algaenan-based wall carries a fibrillar coat not evident in the lichen, and its pyrenoid and plastoglobule endowments are less well developed. These findings are followed by four reports on the QFDEEM ultrastructure of four species of lichens, where we describe the differentiations that occur when the fungi and algae are living together in community with bacteria.

Lichen 3. Outer layers

A lichen is a slow-growing niche-constructing organism that forms a thallus via scripted symbiotic/mutualist relationships between fungi, algae, and bacteria. Here we use quick-freeze deep-etch electron microscopy (QFDEEM), in conjunction with light microscopy, to document the structural manifestations of hyphal differentiation during the formation of three lichen tissues that localize between the algal layer and the surface of the thallus: the outer cortex of foliose lobes; the outer layer of fruticose stems; and the enwrapping layer of asexual propagules called soredia that protrude from squamulose podetia and foliose lobes. Our observations document features of outer-layer architecture and the role played by extracellular matrices (ECM). They also lead us to propose the medullary stem-cell hypothesis for lichen organization wherein totipotent medullary hyphae produce lateral branches that undergo specific differentiation pathways in specific domains of the thallus.

Involvement of a Multidrug Efflux Pump and Alterations in Cell Surface Structure in the Synergistic Antifungal Activity of Nagilactone E and Anethole against Budding Yeast Saccharomyces cerevisiae.

Nagilactone E, an antifungal agent derived from the root bark of Podocarpus nagi, inhibits 1,3-β glucan synthesis; however, its inhibitory activity is weak. Anethole, the principal component of anise oil, enhances the antifungal activity of nagilactone E. We aimed to determine the combinatorial effect and underlying mechanisms of action of nagilactone E and anethole against the budding yeast Saccharomyces cerevisiae. Analyses using gene-deficient strains showed that the multidrug efflux pump PDR5 is associated with nagilactone E resistance; its transcription was gradually restricted in cells treated with the drug combination for a prolonged duration but not in nagilactone-E-treated cells. Green-fluorescent-protein-tagged Pdr5p was intensively expressed and localized on the plasma membrane of nagilactone-E-treated cells but not in drug-combination-treated cells. Quick-freeze deep-etch electron microscopy revealed the smoothening of intertwined fiber structures on the cell surface of drug-combination-treated cells and spheroplasts, indicating a decline in cell wall components and loss of cell wall strength. Anethole enhanced the antifungal activity of nagilactone E by enabling its retention within cells, thereby accelerating cell wall damage. The combination of nagilactone E and anethole can be employed in clinical settings as an antifungal, as well as a food preservative to restrict food spoilage.

Ultrastructure of the green alga Dunaliella salina strain CCAP19/18 (Chlorophyta) as investigated by quick-freeze deep-etch electron microscopy

The single-celled green alga Dunaliella salina is a model system for studies on stress biology, in particular regarding secondary carotenoid accumulation. Under non-stress conditions the cells are green, but under abiotic stress the cells turn orange, because they switch their metabolism and accumulate β-Carotene in globules in the chloroplast. For the first time, Quick-freeze deep-etch electron microscopy was used to visualize cellular structures in green and orange cells of D. salina strain CCAP19/18. This allowed us to present an in-depth analysis of the cellular ultrastructure describing and comparing the features of the two cell types. Our images illustrate the presence of a pericellular matrix for this strain of D. salina. The pericellular matrix was spongy and strands of unknown material anchored it into the plasma membrane. The cytoplasm contained a variety of vesicles, vacuoles, and acidocalcisomes. We could show for strain CCAP19/18 that cytoplasmic lipid bodies were often in close proximity to and sometimes in contact with the outer chloroplast envelope membrane and with the endoplasmic reticulum. Major visible differences between green and orange cells were in the chloroplast: the orange cells have greatly reduced amounts of thylakoid membranes and greatly increased numbers of β-Carotene globules. We showed that the β-Carotene globules often made point contacts with thylakoid membranes, and frequently laid side-by-side along the thylakoid membrane surface, providing support to studies that indicated exchange of molecules between β-Carotene globules and thylakoid membranes. A novel finding was the β-Carotene globule duplets, suggesting intermediate stages in β-Carotene globule morphogenesis. Overall, the β-Carotene globules appear to be similar to plant plastoglobuli. We provide this description of the cellular ultrastructure features as a resource in the context of the recent publication of the genome of D. salina strain CCAP19/18 to expand on the knowledge regarding this novel reference strain.

Analysis of an improved Cyanophora paradoxa genome assembly.

Glaucophyta are members of the Archaeplastida, the founding group of photosynthetic eukaryotes that also includes red algae (Rhodophyta), green algae, and plants (Viridiplantae). Here we present a high-quality assembly, built using long-read sequences, of the ca. 100 Mb nuclear genome of the model glaucophyte Cyanophora paradoxa. We also conducted a quick-freeze deep-etch electron microscopy (QFDEEM) analysis of C. paradoxa cells to investigate glaucophyte morphology in comparison to other organisms. Using the genome data, we generated a resolved 115-taxon eukaryotic tree of life that includes a well-supported, monophyletic Archaeplastida. Analysis of muroplast peptidoglycan (PG) ultrastructure using QFDEEM shows that PG is most dense at the cleavage-furrow. Analysis of the chlamydial contribution to glaucophytes and other Archaeplastida shows that these foreign sequences likely played a key role in anaerobic glycolysis in primordial algae to alleviate ATP starvation under night-time hypoxia. The robust genome assembly of C. paradoxa significantly advances knowledge about this model species and provides a reference for exploring the panoply of traits associated with the anciently diverged glaucophyte lineage.

Quantitative biogeography of picoprasinophytes establishes ecotype distributions and significant contributions to marine phytoplankton.

Bathycoccus and Ostreococcus are broadly distributed marine picoprasinophyte algae. We enumerated small phytoplankton using flow cytometry and qPCR assays for phylogenetically distinct Bathycoccus clades BI and BII and Ostreococcus clades OI and OII. Among 259 photic-zone samples from transects and time-series, Ostreococcus maxima occurred inthe North Pacific coastal upwelling for OI (36713 ± 1485 copiesml-1 ) and the Kuroshio Front for OII (50189 ± 561 copiesml-1 ) and the two overlapped only in frontal regions. The Bathycoccus overlapped more often with maxima along Line-P for BI (10667 ± 1299 copiesml-1 ) and the tropical Atlantic for BII (4125 ± 339 copiesml-1 ). Only BII and OII were detected at warm oligotrophic sites, accounting for 34 ± 13% of 1589 ± 448 eukaryotic phytoplankton cellsml-1 (annual average) at Station ALOHA's deep chlorophyll maximum. Significant distributional and molecular differences lead us to propose that Bathycoccus clade BII represents a separate species which tolerates higher temperature oceanic conditions than Bathycoccus prasinos (BI). Morphological differences were not evident, but quick-freeze deep-etch electron microscopy provided insight into Bathycoccus scale formation. Our results highlight the importance of quantitative seasonal abundance data for inferring ecological distributions and demonstrate significant, differential picoprasinophyte contributions in mesotrophic and open-ocean waters.

Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens.

Eisosomes are among the few remaining eukaryotic cellular differentations that lack a defined function(s). These trough-shaped invaginations of the plasma membrane have largely been studied in Saccharomyces cerevisiae, in which their associated proteins, including two BAR domain proteins, have been identified, and homologues have been found throughout the fungal radiation. Using quick-freeze deep-etch electron microscopy to generate high-resolution replicas of membrane fracture faces without the use of chemical fixation, we report that eisosomes are also present in a subset of red and green microalgae as well as in the cysts of the ciliate Euplotes. Eisosome assembly is closely correlated with both the presence and the nature of cell walls. Microalgal eisosomes vary extensively in topology and internal organization. Unlike fungi, their convex fracture faces can carry lineage-specific arrays of intramembranous particles, and their concave fracture faces usually display fine striations, also seen in fungi, that are pitched at lineage-specific angles and, in some cases, adopt a broad-banded patterning. The conserved genes that encode fungal eisosome-associated proteins are not found in sequenced algal genomes, but we identified genes encoding two algal lineage-specific families of predicted BAR domain proteins, called Green-BAR and Red-BAR, that are candidate eisosome organizers. We propose a model for eisosome formation wherein (i) positively charged recognition patches first establish contact with target membrane regions and (ii) a (partial) unwinding of the coiled-coil conformation of the BAR domains then allows interactions between the hydrophobic faces of their amphipathic helices and the lipid phase of the inner membrane leaflet, generating the striated patterns.

3-D structural analysis of the crucial intermediate of skeletal muscle myosin and its role in revised actomyosin cross-bridge cycle.

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.

Novel configuration of a myosin II transient intermediate analogue revealed by quick-freeze deep-etch replica electron microscopy

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.

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.

Fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy

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 (D(WP)) and SP (D(SP)). The result indicates that D(WP) was larger than D(SP) 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.

Analysis of Conformation of the Skeletal Muscle Myosin Modified by F2DNB Using FRET

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 skeletal muscle heavy mero-myosin (HMM), which SH1-SH2 was crosslinked by 1,5-difluoro-2, 4-dinitrobenzene (F2DNB) 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 F2DNB using FRET between the ATP binding site and the essential light chain (ELC) A1. We prepared 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 within 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. The FRET efficiency of F2DNB-S1-NBD-ADP was lower than S1-NBD-ADP state. This suggests that the F2DNB-S1-ADP states form more kinked conformation than S1-ADP state.

Dynamic shear-influenced collagen self-assembly

The ability to influence the direction of polymerization of a self-assembling biomolecular system has the potential to generate materials with extremely high anisotropy. In biological systems where highly-oriented cellular populations give rise to aligned and often load-bearing tissue such organized molecular scaffolds could aid in the contact guidance of cells for engineered tissue constructs (e.g. cornea and tendon). In this investigation we examine the detailed dynamics of pepsin-extracted type I bovine collagen assembly on a glass surface under the influence of flow between two plates. Differential Interference Contrast (DIC) imaging (60×-1.4NA) with focal plane stabilization was used to resolve and track the growth of collagen aggregates on borosilicate glass for 4 different shear rates (500, 80, 20, and 9 s −1). The detailed morphology of the collagen fibrils/aggregates was examined using Quick Freeze Deep Etch (QFDE) electron microscopy. Nucleation of fibrils on the glass was observed to occur rapidly (∼2 min) followed by continued growth of the fibrils. The growth rates were dependent on flow in a complex manner with the highest rate of axial growth (0.1 μ/s) occurring at a shear rate of 9 s −1. The lowest growth rate occurred at the highest shear. Fibrils were observed to both branch and join during the experiments. The best alignment of fibrils was observed at intermediate shear rates of 20 and 80 s −1. However, the investigation revealed that fibril directional growth was not stable. At high shear rates, fibrils would often turn downstream forming what we term “hooks” which are likely the combined result of monomer interaction with the initial collagen layer or “mat” and the high shear rate. Further, QFDE examination of fibril morphology demonstrated that the assembled fibrillar structure did not possess native D-periodicity. Instead, fibrils comprised a collection of generally aligned, monomers which were self-assembled to form a fibril-like aggregate. In conclusion, though constant shear-rate clearly influences collagen fibrillar alignment, the formation of highly-organized collagenous arrays of native-like D-banded fibrils remains a challenge. Modulation of shear in combination with surface energy patterning to produce a highly-aligned initial mat may provide significant improvement of both the fibril morphology and alignment.

Analysis of Conformation of Skeletal Muscle Myosin Cross-linked by pPDM Using FRET

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.