Journal of Structural Biology: X最新文献

AreTomo, an abbreviation for Alignment and Reconstruction for Electron Tomography, is a GPU accelerated software package that fully automates motion-corrected marker-free tomographic alignment and reconstruction in a single package. By correcting in-plane rotation, translation, and importantly, the local motion resulting from beam-induced motion from tilt to tilt, AreTomo can produce tomograms with sufficient accuracy to be directly used for subtomogram averaging. Another major application is the on-the-fly reconstruction of tomograms in parallel with tilt series collection to provide users with real-time feedback of sample quality allowing users to make any necessary adjustments of collection parameters. Here, the multiple alignment algorithms implemented in AreTomo are described and the local motions measured on a typical tilt series are analyzed. The residual local motion after correction for global motion was found in the range of ± 80 Å, indicating that the accurate correction of local motion is critical for high-resolution cryo-electron tomography (cryoET).

Cryo-electron tomography (Cryo-ET) provides unique opportunities to image cellular components at high resolution in their native state and environment. While many different cell types were investigated by cryo-ET, here we review application to neurons. We show that neurons are a versatile system that can be used to investigate general cellular components such as the cytoskeleton and membrane-bound organelles, in addition to neuron-specific processes such as synaptic transmission. Furthermore, the synapse provides a rich environment for the development of cryo-ET image processing tools suitable to elucidate the functional and spatial organization of compositionally and morphologically heterogeneous macromolecular complexes involved in biochemical signaling cascades, within their native, crowded cellular environments.

The resolution of cryo-EM reconstructions is fundamentally limited by the Nyquist frequency, which is half the sampling frequency of the detector and depends upon the magnification used. In principle, super-resolution imaging should enable reconstructions to surpass the physical Nyquist limit by increasing sampling frequency, yet there are few reports of reconstructions that do so. Here we directly examine the contribution of super-resolution information, obtained with the K3 direct electron detector using a 2-condenser microscope, to single-particle cryo-EM reconstructions surpassing the physical Nyquist limit. We also present a comparative analysis of a sample imaged at four different magnifications. This analysis demonstrates that lower magnifications can be beneficial, despite the loss of higher resolution signal, due to the increased number of particle images obtained. To highlight the potential utility of lower magnification data collection, we produced a 3.5 Å reconstruction of jack bean urease with particles from a single micrograph.

Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonas l-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Å resolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing.

The roles of ISL1 and LHX3 in the development of spinal motor neurons have been well established. Whereas LHX3 triggers differentiation into interneurons, the additional expression of ISL1 in developing neuronal cells is sufficient to redirect their developmental trajectory towards spinal motor neurons. However, the underlying mechanism of this action by these transcription factors is less well understood. Here, we used electrophoretic mobility shift assays (EMSAs) and surface plasmon resonance (SPR) to probe the different DNA-binding behaviours of these two proteins, both alone and in complexes mimicking those found in developing neurons, and found that ISL1 shows markedly different binding properties to LHX3. We used small angle X-ray scattering (SAXS) to structurally characterise DNA-bound species containing ISL1 and LHX3. Taken together, these results have allowed us to develop a model of how these two DNA-binding modules coordinate to regulate gene expression and direct development of spinal motor neurons.

Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 h. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy.

Among the terrestrial Crustacea, isopods have most successfully established themselves in a large variety of terrestrial habitats. As in most Crustacea, their cuticle consists of a hierarchically organised organic phase of chitin-protein fibrils, containing calcium carbonate and some calcium phosphate. In previous studies, we examined the tergite cuticle of Tylos europaeus, which lives on seashores and burrows into moist sand. In this study, we investigate the closely related species Helleria brevicornis, which is completely terrestrial and lives in leaf litter and humus and burrows into the soil. To get deeper insights in relation between the structure of the organic and mineral phase in species living in diverse habitats, we have investigated the structure, and the chemical and crystallographic properties of the tergite cuticle using various preparation techniques, and microscopic and analytical methods. The results reveal long and short epicuticular sensilla with brushed tips on the tergite surface that do not occur in T. europaeus. As in T. europaeus a distal exocuticle, which contains a low number of organic fibres, contains calcite while the subjacent layers of the exo- and endocuticle contain amorphous calcium carbonate. The distal exocuticle contains a polygonal pattern of mineral initiation sites that correspond to interprismatic septa described for decapod crabs. The shape and position of calcite units do not follow the polygonal pattern of the septa. The results indicate that the calcite units form by crystallisation from an amorphous phase that progresses from both margins of the septa to the centres of the polygons.

Biomineralization is the process of mineral formation by living organisms. One notable example of these organisms is magnetotactic bacteria (MTB). MTB are Gram-negative bacteria that can biomineralize iron into magnetic nanoparticles. This ability allows these aquatic microorganisms to orient themselves according to the geomagnetic field. The biomineralization process takes place in a specialized sub-cellular membranous organelle, the magnetosome. The magnetosome contains a defined set of magnetosome-associated proteins (MAPs) that controls the biomineralization environment, including iron concentration, redox, and pH. Magnetite formation is subjected to a tight regulation within the magnetosome that affects the nanoparticle nucleation, size, and shape, leading to well-defined magnetic properties. The formed magnetite nanoparticles have unique characteristics of a stable, single magnetic domain with narrow size distribution and high crystalline structures, which turned MTB into the subject of interest in multidisciplinary research. This graphical review provides a current overview of iron biomineralization in magnetotactic bacteria, focusing on Alphaproteobacteria. To better understand this complex mechanism, we present the four main steps and the main MAPs participating in the process of magnetosome formation.

The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-β-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-β-1,4-mannosidase [EC 3.2.1.25] that is highly specific for β-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Å resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family β-galactosidases and the GH164-family exo-β-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 β-galactosidase-type homotrimeric structure.

Mechanosensitive (MS) channels that are activated by the ‘force-from-lipids’ (FFL) principle rest in the membrane in a closed state but open a transmembrane pore in response to changes in the transmembrane pressure profile. The molecular implementations of the FFL principle vary widely between different MS channel families. The function of MS channels is often studied by patch-clamp electrophysiology, in which mechanical force or amphipathic molecules are used to activate the channels. Structural studies of MS channels in states other than the closed resting state typically relied on the use of mutant channels. Cyclodextrins (CDs) were recently introduced as a relatively easy and convenient approach to generate membrane tension. The principle is that CDs chelate hydrophobic molecules and can remove lipids from membranes, thus forcing the remaining lipids to cover more surface area and creating tension for membrane proteins residing in the membranes. CDs can be used to study the structure of MS channels in a membrane under tension by using single-particle cryo-electron microscopy to image the channels in nanodiscs after incubation with CDs as well as to characterize the function of MS channels by using patch-clamp electrophysiology to record the effect of CDs on channels inserted into membrane patches excised from proteoliposomes. Importantly, because incubation of membrane patches with CDs results in the activation of MscL, an MS channel that opens only shortly before membrane rupture, CD-mediated lipid removal appears to generate sufficient force to open most if not all types of MS channels that follow the FFL principle.