3,4-Dihydroxybenzalacetone Inhibits the Propagation of Hydrogen Peroxide-Induced Oxidative Effect via Secretory Components from SH-SY5Y Cells.

The polyphenol derivative 3,4-dihydroxybenzalacetone (DBL) is the primary antioxidative component of the medicinal folk mushroom Chaga (Inonotus obliquus (persoon) Pilat). In this study, we investigated whether the antioxidative effect of DBL could propagate to recipient cells via secreted components, including extracellular vesicles (EVs), after pre-exposing SH-SY5Y human neuroblastoma cells to DBL. First, we prepared EV-enriched fractions via sucrose density gradient ultracentrifugation using conditioned medium from SH-SY5Y cells exposed to 100 µM hydrogen peroxide (H2O2) for 24 h, with and without 1 h of 5 µM DBL pre-treatment. CD63 immuno-dot blot analysis demonstrated that fractions with density of 1.06-1.09 g/cm3 had CD63-like immuno-reactivities. Furthermore, the 2,2-diphenyl-1-picrylhydrazyl assay revealed that the radical scavenging activity of fraction 11 (density of 1.06 g/cm3), prepared after 24-h H2O2 treatment, was significantly increased compared to that in the control group (no H2O2 treatment). Notably, 1 h of 5 µM DBL pre-treatment or 5 min of heat treatment (100 °C) diminished this effect, although concentrating the fraction by 100 kDa ultrafiltration enhanced it. Overall, the effect was not specific to the recipient cell types. In addition, the uptake of fluorescent Paul Karl Horan-labeled EVs in concentrated fraction 11 was detected in all treatment groups, particularly in the H2O2-treated group. The results suggest that cell-to-cell communication via bioactive substances, such as EVs, in conditioned SH-SY5Y cell medium, propagates the H2O2-induced radical scavenging effect, whereas pre-conditioning with DBL inhibits it.

Investigation of the Efficacy of Lanthanoid Heavy Metal Acetates as Electron Staining Reagents for Biomembrane Vesicles.

Transmission electron microscopy (TEM) has revolutionized our understanding of protein structures by enabling atomic-resolution visualization without the need for crystallography, thanks to advancements in cryo-TEM and single particle analysis methods. However, conventional electron microscopy remains relevant for studying stained samples, as it allows the practical determination of optimal conditions through extensive experimentation. TEM also facilitates the examination of supramolecular complexes encompassing proteins, lipids, and nucleic acids. In this study, we investigated the applicability of lanthanoid reagents as electron-staining alternatives to uranyl acetate, which is globally regulated as a nuclear fuel material. We focus on a model biomembrane vesicle system, the chromatophores from the purple photosynthetic eubacterium Rhodospirillum rubrum, which integrate proteins and lipids. Through density distribution analysis of electron micrographs, we evaluated the efficacy of various lanthanoid acetates and found that triacetates of neodymium, samarium, and gadolinium exhibited similar staining effectiveness to uranyl acetate. Additionally, triacetates of praseodymium, erbium, and lutetium, followed by europium show promising results as secondary candidates. Our findings suggest that lanthanoid transition heavy metal acetates could serve as viable alternatives for electron staining in TEM, offering potential advantages over uranyl acetate.

Optimizing Exosome Preparation Based on Size and Morphology: Insights From Electron Microscopy.

Extracellular vesicles (EVs), including exosomes, are crucial in intercellular communication, but differentiating between exosomes and microvesicles is challenging due to their similar morphology and size. This study focuses on multivesicular bodies (MVBs), where exosomes mature, and optimizes exosome isolation using transmission electron microscopy (TEM) for size information. Considering that EVs are nanocolloidal particles, a salt-free Bis-Tris buffer is found to maintain EV integrity better than phosphate-buffered saline (PBS). Dynamic light scattering (DLS) and TEM analysis confirm that intact exosome fractions under the salt-free Bis-Tris buffer condition exhibit polydispersity, including a unique population of <50 nm vesicles resembling intraluminal membrane vesicles (ILVs) in MVBs, alongside larger populations. This <50 nm population disappears in PBS or Bis-Tris buffer with 140 mM NaCl, transforming into a monodisperse population >100 nm. Immunoelectron microscopy also validates the presence of CD63, an exosome biomarker, on approximately 50 nm EVs. These findings provide valuable insights into exosome characterization and isolation, essential for future biomedical applications in diagnostics and drug delivery.

Systematic Investigation of Lanthanoid Transition Heavy Metal Acetates as Electron Staining Reagents for Protein Molecules in Biological Transmission Electron Microscopy.

Cryo-electron microscopy, widely used for high-resolution protein structure determination, does not require staining. Yet negative staining with heavy metal salts such as uranyl acetate has been in persistent demand since the 1950s due to its image contrasting capabilities at room temperature with a common electron microscope. However, uranium compounds are nuclear fuel materials and are tightly controlled worldwide. Acetates of each lanthanoid series elements except promethium are prepared at the same concentration (2%(w/v)) and used as a model on horse spleen ferritin for electron microscopic analysis to systematically evaluate their effectiveness as electron staining reagents for the protein. Analysis shows that the triacetates of samarium and europium, followed by gadolinium and erbium, and then lanthanum and neodymium could function as electron staining reagents. Thulium-triacetate precipitates thin plate-like crystals and may be used for selecting better imaging fields. Of the 14 lanthanoid-triacetates examined, about half are viable alternatives to uranyl acetate as an electron staining reagent for ferritin, and there appears an optimal range in ionic sizes for promising lanthanoids. This is the first systematic investigation of lanthanoid transition heavy metal triacetates from the viewpoint of lanthanoid contraction, using density distribution histograms of electron micrographs as an indicator for comparison with uranyl acetate.

GroEL and the GroEL-GroES Complex.

Chaperonin is categorized as a molecular chaperone and mediates the formation of the native conformation of proteins by first preventing folding during synthesis or membrane translocation and subsequently by mediating the step-wise ATP-dependent release that result in proper folding. In the GroEL-GroES complex, a single heptameric GroEL ring binds one GroES ring in the presence of ATP/ADP, in this vein, the double ring GroEL tetradecamer is present in two distinct types of GroEL-GroES complexes: asymmetric 1:1 "bullet"-shaped GroEL:GroES and symmetric 1:2 "football" (American football)-shaped GroEL:GroES2. There have been debates as to which complex is critical to the productive protein folding mediated by the GroEL-GroES complex, and how GroES coordinates with GroEL in the chaperonin reaction cycle in association with regulation by adenine nucleotides and through the interplay of substrate proteins. A lot of knowledge on chaperonins has been accumulating as if expanding as ripples spread around the GroEL-GroES from Escherichia coli. In this article, an overview is presented on GroEL and the GroEL-GroES complex, with emphasis on their morphological variations, and some potential applications to the fabrication of nanocomposites using GroEL as a nano-block. In parallel, a guideline is presented that supports the recognition that the E. coli and its GroEL-GroES complex do not always receive in standard literature because the biochemical features of chaperonins derived from others special, such as mammals, are not always the same as those confirmed using GroEL-GroES derived from E. coli.

Anisotropic intersubunit and inter-ring interactions revealed in the native bullet-shaped chaperonin complex from Thermus thermophilus.

BACKGROUND: The recent morphological studies on chaperonins have revealed that nearly equivalent amount of symmetric GroEL-(GroES)2 (football-shaped) and asymmetric GroEL-GroES (bullet-shaped) complexes coexist during the chaperonin reaction cycle, which prompted us to reexamine the equatorial split observed for chaperonin from Thermus thermophilus by implementing semi-empirical molecular orbital (MO) calculations, since it is now believed that the symmetric formation is a precursor to the equatorial split. METHODS: Semi-empirical MO calculations were employed to investigate the intersubunit interactions within the bullet-shaped T. thermophilus chaperonin capturing the substrate of folding intermediates. Interaction energies between each cis-GroEL subunit and closely related remaining subunits in cis-GroEL ring, or in trans-GroEL ring across the equatorial plane, and further, interaction energies between each GroES subunit and adjacent subunits in the same GroES ring and in cis-GroEL ring were simulated. RESULTS: Anisotropic intensities and energy distribution of the subunits were revealed by the calculations, which are consistent with two conformations of the subunits forming cis-GroEL ring as revealed by X-ray crystal structure, and with an anisotropic critical binding site on cis-GroEL ring for chaperonin functioning. CONCLUSIONS: This is the first application of semi-empirical MO calculations to the macromolecular complex of the native bullet-shaped chaperonin (GroEL-GroES-ADP homolog) from T. thermophilus. GENERAL SIGNIFICANCE: The results also appear to support the occurrence of the equatorial split for T. thermophilus chaperonin observed via electron microscopy, but has not yet been fully observed for Escherichia coli GroEL-GroES system.

Dynamic or nondynamic? Helical trajectory in hexabenzocoronene nanotubes biased by a detachable chiral auxiliary.

When ether vapor was allowed to diffuse into a CH(2)Cl(2) solution of an enantiomer of a hexa-peri-hexabenzocoronene (HBC) derivative carrying a chiral (BINAP)Pt(II)-appended coordination metallacycle (HBC(Py)([(R)-Pt]) or HBC(Py)([(S)-Pt])), screw-sense-selective assembly took place to give optically active nanotubes (NT(Py)([(R)-Pt]) or NT(Py)([(S)-Pt])) with helical chirality, which were enriched in either left-handed (M)-NT(Py)([(R)-Pt]) or right-handed (P)-NT(Py)([(S)-Pt]), depending on the absolute configuration of the (BINAP)Pt(II) pendant. When MeOH was used instead of ether for the vapor-diffusion-induced assembly, nanocoils formed along with the nanotubes. As determined by scanning electron microscopy, the diastereomeric excess of the nanocoils was 60% (80:20 diastereomeric ratio). Removal of the (BINAP)Pt(II) pendants from NT(Py)([(R)-Pt]) or NT(Py)([(S)-Pt]) with ethylenediamine yielded metal-free nanotubes (NT(Py)) that remained optically active even upon heating without any change in the circular dichroism spectral profile. No helical inversion took place when NT(Py) derived from HBC(Py)([(R)-Pt]) or HBC(Py)([(S)-Pt]) was allowed to complex with (BINAP)Pt(II) with an absolute configuration opposite to the original one.

Two-dimensional crystalline array formation of glucuronide transporter from Escherichia coli by the use of polystyrene beads for detergent removal.

n-Dodecyl-ß-D-maltoside solubilized glucuronide transporter (GusB), the product of gusB gene from Escherichia coli, was treated with Bio-Beads as an agent for removing the detergent from a micellar solution under suitable combination with dimyristoylphosphatidylcholine. Optimizing conditions led to a two-dimensional crystalline array formation of GusB. The crystalline arrays appear to have a hexagonal lattice with layer group P6, the unit cell dimensions of a = b = 13.8 nm and γ = 120°. Each stain-protruding periodic unit showed approximately 11.8 ± 0.3 nm in a diameter in the inverse Fourier-filtered image to have formed with pentameric GusB (5 × 49.7 kDa).

C-shaped dipper: A novel useful auxiliary tool for preparation of specimen grids for transmission electron microscopy.

The transmission electron microscopy (TEM) has played a pivotal role in visualizing and detailing the morphology of nanoscale objects of observation, but care must be taken to ensure that the object is in its original, unperturbed state. This is because not all objects under the observation are necessarily robust and structurally stable, and if the object being observed is so delicate and easily changeable in shape, one may be observing structural artifacts leading to wrong interpretations. I here present a novel prototype of an easy-to-use and reliable auxiliary tool, the C-shaped dipper, developed for the preparation of specimen grids for TEM, along with its handling instructions for diverse applications. Some nanostructures are so delicate and fragile that their nanocomplexes are easily destroyed when aspirated by a pipette with a narrow tip diameter. Using the C-shaped dipper, the observation objects can be scooped up in a droplet formed by the surface tension of the liquid, keeping their intact shapes, and placed on a grid precovered with a support film. The C-shaped dipper has proven to be excellent for handling soft assemblies having delicate structures, such as nano-scale composites, for facilitating thin layered formations with molecular constituent blocks, and for preparing a grid individually covered with a collodion film or an amorphous carbon support film.

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions.

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein-lipid specificity between the two chromatophores.

Ambipolar-transporting coaxial nanotubes with a tailored molecular graphene-fullerene heterojunction.

Despite a large steric bulk of C(60), a molecular graphene with a covalently linked C(60) pendant [hexabenzocoronene (HBC)-C(60); 1] self-assembles into a coaxial nanotube whose wall consists of a graphite-like pi-stacked HBC array, whereas the nanotube surface is fully covered by a molecular layer of clustering C(60). Because of this explicit coaxial configuration, the nanotube exhibits an ambipolar character in the field-effect transistor output [hole mobility (micro(h)) = 9.7 x 10(-7) cm(2) V(-1) s(-1); electron mobility (micro(e)) = 1.1 x 10(-5) cm(2) V(-1) s(-1)] and displays a photovoltaic response upon light illumination. Successful coassembly of 1 and an HBC derivative without C(60) (2) allows for tailoring the p/n heterojunction in the nanotube, so that its ambipolar carrier transport property can be optimized for enhancing the open-circuit voltage in the photovoltaic output. As evaluated by an electrodeless method called flash-photolysis time-resolved microwave conductivity technique, the intratubular hole mobility (2.0 cm(2) V(-1) s(-1)) of a coassembled nanotube containing 10 mol % of HBC-C(60) (1) is as large as the intersheet mobility in graphite. The homotropic nanotube of 2 blended with a soluble C(60) derivative [(6,6)-phenyl C(61) butyric acid methyl ester] displayed a photovoltaic response with a much different composition dependency, where the largest open-circuit voltage attained was obviously lower than that realized by the coassembly of 1 and 2.

Investigation on stability of transporter protein, glucuronide transporter from Escherichia coli.

The glucuronide transporter GusB, the product of the gusB gene from Escherichia coli, is responsible for detoxification of metabolites. In this study, we successfully expressed GusB homologously in E. coli and investigated its oligomeric state in n-dodecyl-beta-D: -maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 +/- 2 A for the purified GusB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GusB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GusB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GusB with lipids.

Novel Caffeic Acid Phenethyl Ester-Mortalin Antibody Nanoparticles Offer Enhanced Selective Cytotoxicity to Cancer Cells.

Caffeic acid phenethyl ester (CAPE) is a key bioactive ingredient of honeybee propolis and is claimed to have anticancer activity. Since mortalin, a hsp70 chaperone, is enriched in a cancerous cell surface, we recruited a unique cell internalizing anti-mortalin antibody (MotAb) to generate mortalin-targeting CAPE nanoparticles (CAPE-MotAb). Biophysical and biomolecular analyses revealed enhanced anticancer activity of CAPE-MotAb both in in vitro and in vivo assays. We demonstrate that CAPE-MotAb cause a stronger dose-dependent growth arrest/apoptosis of cancer cells through the downregulation of Cyclin D1-CDK4, phospho-Rb, PARP-1, and anti-apoptotic protein Bcl2. Concomitantly, a significant increase in the expression of p53, p21WAF1, and caspase cleavage was obtained only in CAPE-MotAb treated cells. We also demonstrate that CAPE-MotAb caused a remarkably enhanced downregulation of proteins critically involved in cell migration. In vivo tumor growth assays for subcutaneous xenografts in nude mice also revealed a significantly enhanced suppression of tumor growth in the treated group suggesting that these novel CAPE-MotAb nanoparticles may serve as a potent anticancer nanomedicine.

Characterization of a sHsp of Schizosaccharomyces pombe, SpHsp15.8, and the implication of its functional mechanism by comparison with another sHsp, SpHsp16.0.

There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citrate synthase (CS) from porcine heart and dithiothreitol-induced aggregation of insulin from bovine pancreas with very high efficiency. Almost one SpHsp15.8 subunit was sufficient to protect one protein molecule from aggregation. Like SpHsp16.0, SpHsp15.8 dissociated into small oligomers and then interacted with denatured substrate proteins. SpHsp16.0 exhibited a clear enthalpy change for denaturation occurring over 60 degrees C in differential scanning calorimetry (DSC). However, we could not observe any significant enthalpy change in the DSC of SpHsp15.8. The difference is likely to be caused by the adhesive characteristics of SpHsp15.8. The oligomer dissociation of SpHsp15.8 and SpHsp16.0 and their interactions with denatured substrate proteins were studied by fluorescence polarization analysis (FPA). Both sHsps exhibited a temperature-dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of the SpHsp15.8 oligomer began at about 35 degrees C and proceeded gradually. On the contrary, the SpHsp16.0 oligomer was stable up to approximately 45 degrees C, but then dissociated into small oligomers abruptly at this temperature. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, while SpHsp15.8 is likely to interact with CS in a large complex. These results suggest that S. pombe utilizes two sHsps that function in different manners, probably to cope with a wide range of temperatures and various denatured proteins.

Molecular glues carrying multiple guanidinium ion pendants via an oligoether spacer: stabilization of microtubules against depolymerization.

Dendron G1(Gu(+))(9)R and linear peptide oligomer Asn(TEG-Gu(+))(9), decorated with multiple guanidinium (Gu(+)) ions as sticky pendants via an oligo(oxyethylene) spacer, adhere to BSA and protein assemblies such as microtubules in aqueous buffers. Using fluorescently labeled G1(Gu(+))(9)R with pyrenyl and rhodamine focal cores, the adhesion process can be visualized by FRET or confocal laser scanning microscopy. The adhesion to microtubules leads to their stabilization against depolymerization into alpha/beta-tubulin heterodimer components, where the effects of G1(Gu(+))(9)R and Asn(TEG-Gu(+))(9) are comparable to that of paclitaxel, known as an anticancer drug. Since G1(Gu(+))(9)R and Asn(TEG-Gu(+))(9) are superior to lower-generation G0(Gu(+))(3)OMe and arginine nonamer, respectively, the multivalency of the interaction and a conformational flexibility of the oligoether spacers play a crucial role in the efficient adhesion to proteins.

A tubular biocontainer: metal ion-induced 1D assembly of a molecularly engineered chaperonin.

GroEL(SP/MC), prepared by genetic and chemical modifications of group I chaperonin protein GroEL, site-specifically possesses approximately 28 photochromic (spiropyran [SP] and merocyanine [MC]) units in the entrance parts of its cavity. Addition of divalent metal ions such as Mg(2+) to a tris-HCl buffer solution of GroEL(SP/MC) results in one-dimensional (1D) assembly of GroEL(SP/MC), affording cylindrical hollow fibers with a very large aspect ratio; the longest fiber was approximately 2.5 microm long, corresponding to a 170-mer of GroEL(SP/MC) (MW approximately 1.4 x 10(8)). When such long fibers are mixed with EDTA, they are cut into short-chain oligomers and eventually into monomeric GroEL(SP/MC). Similar to GroEL, GroEL(SP/MC) possesses a large binding affinity toward denatured proteins. When GroEL(SP/MC) undergoes 1D assembly after incubation with a denatured protein, guest-containing cylindrical fibers result.

Metal-ion-coated graphitic nanotubes: controlled self-assembly of a pyridyl-appended gemini-shaped hexabenzocoronene amphiphile.

The assembly line: Hexabenzocoronene amphiphiles appended with pyridyl-terminated triethylene glycol side chains, in combination with trans-[Pt(PhCN)(2)Cl(2)], lead to the formation of graphitic nanotubes. The structural features and dimensions of the nanotubes depend on the assembly conditions. A platinum(II)-bridged cyclic dimer having two HBC units self-assembles into a nanotubular structure.

Role of the IXI/V motif in oligomer assembly and function of StHsp14.0, a small heat shock protein from the acidothermophilic archaeon, Sulfolobus tokodaii strain 7.

Small heat shock proteins (sHsps) are one of the most ubiquitous molecular chaperones. They are grouped together based on a conserved domain, the alpha-crystallin domain. Generally, sHsps exist as oligomers of 9-40 subunits, and the oligomers undergo reversible temperature-dependent dissociation into smaller species as dimers, which interact with denaturing substrate proteins. Previous studies have shown that the C-terminal region, especially the consensus IXI/V motif, is responsible for oligomer assembly. In this study, we examined deletions or mutations in the C-terminal region on the oligomer assembly and function of StHsp14.0, an sHsp from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Mutated StHsp14.0 with C-terminal deletion or replacement of IIe residues in the IXI/V motif to Ala, Ser, or Phe residues could not form large oligomers and lost chaperone activity. StHsp14.0WKW, whose Ile residues in the IXI/V motif are changed to Trp, existed as an oligomer like that of the wild type. However, it dissociates to small oligomers and exhibits chaperone activity at relatively lowered temperature. Replacement of two Ile residues in the motif to relatively small residues, Ala or Ser, also resulted in the change of beta-sheet rich secondary structure and decrease of hydrophobicity. Interestingly, StHsp14.0 mutant with amino acid replacements to Phe kept almost the same secondary structure and relatively high hydrophobicity despite that it could not form an oligomeric structure. The results show that hydrophobicity and size of the amino acids in the IXI/V motif in the C-terminal region are responsible not only for assembly of the oligomer but also for the maintenance of beta-sheet rich secondary structure and hydrophobicity, which are important for the function of sHsp.

Systematic studies on structural parameters for nanotubular assembly of hexa-peri-hexabenzocoronenes.

Thirteen different hexa-peri-hexabenzocoronenes (HBCs) I-III were newly synthesized, and their self-assembling behaviors were investigated. Taking into account also the reported behaviors of amphiphilic HBCs, some structural parameters of HBC essential for the tubular assembly were revealed. Points to highlight include (1) the importance of two phenyl groups attached to one side of the HBC unit, (2) essential roles of long paraffinic side chains on the other side of the phenyl groups, and (3) no necessity of hydrophilic oligo(ethylene glycol) side chains. The hierarchical nanotubular structure, rendered by virtue of a synchrotron radiation technique, was virtually identical to our previous proposal, where the nanotubes are composed of helically coiled bilayer tapes with a tilting angle of approximately 45 degrees. Each tape consists of pi-stacked HBC units, where the inner and outer HBC layers are connected by interdigitation of paraffinic side chains. The coiled structure is most likely caused by a steric congestion of the phenyl groups attached to the HBC unit, whose tilting direction may determine the handedness of the helically chiral nanotube.

Supramolecular complex formation and crystallization of isocitrate dehydrogenase from Thermus thermophilus HB8: preliminary studies with X-Ray crystallography and atomic force microscopy.

Atomic force microscopy (AFM) observation of a crystal surface of the thermostable isocitrate dehydrogenase (ICDH) from a thermophilic eubacterium, Thermus thermophilus HB8, suggested that the crystal consists of huge homo-complexed ellipsoidal bodies of the protein, with averaged long- and short-axis diameters of 18.6 nm and 10.9 nm respectively. Thick diamond-shaped crystals of about 0.4 mm on the longest axis were obtained by the vapor diffusion method from a solution of 100 mM sodium cacodylate, pH 6.6-8.4, containing 1.4 M sodium acetate as the precipitate, and diffracted X-rays at 3.7 A resolution. The crystals belonged to the monoclinic lattice type with space group C2 and had cell dimensions of a=495.5, b=189.2, c=336.2 A, and beta=126.4 degrees , indicating that an asymmetric unit contained more than 33 molecules with a molecular mass of 54.2 kDa. Calculations based on data obtained by the X-ray method showed good agreement with AFM observation. These results suggest the possibility that the residing T. thermophilus HB8 ICDH molecules are piled one on top another as a preformed supramolecular nano-architecture in the crystal lattice. The system appears suitable for further investigation using a bottom-up approach to the self-associated construction of nano-architectures with proteins.