Journal of Structural Biology: X最新文献

The thermotoga maritima arginine binding protein (TmArgBP) is a periplasmic binding protein that has a short helix at the C-terminal end (CTH), which is swapped between the two chains. We apply a coarse-grained structure-based model (SBM) and all-atom MD simulation on this protein to understand the mechanism and the role of CTH in the conformational transition. When the results of SBM simulations of TmArgBP in the presence and absence of CTH are compared, we find that CTH is strategically located at the back of the binding pocket restraining the open-state conformation thereby disengaging access to the closed-state. We also ran all-atom MD simulations of open-state TmArgBP with and without CTH and discovered that in the absence of CTH the protein could reach the closed-state within 250 ns, while in its presence, the protein remained predominantly in its open-state conformation. In the simulation started from unliganded closed-state conformation without CTH, the protein exhibited multiple transitions between the two states, suggesting CTH as an essential structural element to stabilize the open-state conformation. In another simulation that began with an unliganded closed-state conformation with CTH, the protein was able to access the open-state. In this simulation the CTH was observed to reorient itself to interact with the protein emphasizing its role in assisting the conformational change. Based on our findings, we believe that CTH not only acts as a structural element that constraints the protein in its open-state but it may also guide the protein back to its open-state conformation upon ligand unbinding.

AA amyloidosis is one of the most prevalent forms of systemic amyloidosis and affects both humans and other vertebrates. In this study, we compare MAS solid-state NMR data with a recent cryo-EM study of fibrils involving full-length murine SAA1.1. We address the question whether the specific requirements for the reconstitution of an amyloid fibril structure by cryo-EM can potentially yield a bias towards a particular fibril polymorph. We employ fibril seeds extracted from in to vivo material to imprint the fibril structure onto the biochemically produced protein. Sequential assignments yield the secondary structure elements in the fibril state. Long-range DARR and PAR experiments confirm largely the topology observed in the ex-vivo cryo-EM study. We find that the β-sheets identified in the NMR experiments are similar to the β-sheets found in the cryo-EM study, with the exception of amino acids 33–42. These residues cannot be assigned by solid-state NMR, while they adopt a stable β-sheet in the cryo-EM structure. We suggest that the differences between MAS solid-state NMR and cryo-EM data are a consequence of a second conformer involving residues 33–42. Moreover, we were able to characterize the dynamic C-terminal tail of SAA in the fibril state. The C-terminus is flexible, remains detached from the fibrils, and does not affect the SAA fibril structure as confirmed further by molecular dynamics simulations. As the C-terminus can potentially interact with other cellular components, binding to cellular targets can affect its accessibility for protease digestion.

Fungal infections cause high mortality in immunocompromised individuals, which has emerged as a significant threat to human health. The efforts devoted to the development of antifungal agents targeting the cell wall polysaccharides have been hindered by our incomplete picture of the assembly and remodeling of fungal cell walls. High-resolution solid-state nuclear magnetic resonance (ss NMR) studies have substantially revised our understanding of the polymorphic structure of polysaccharides and the nanoscale organization of cell walls in Aspergillus fumigatus and multiple other fungi. However, this approach requires ¹³C/¹⁵N-enrichment of the sample being studied, severely restricting its application. Here we employ the dynamic nuclear polarization (DNP) technique to compare the unlabeled cell wall materials of A. fumigatus and C. albicans prepared using both liquid and solid media. For each fungus, we have identified a highly conserved carbohydrate core for the cell walls of conidia and mycelia, and from liquid and solid cultures. Using samples prepared in different media, the recently identified function of α-glucan, which packs with chitin to form the mechanical centers, has been confirmed through conventional ss NMR measurements of polymer dynamics. These timely efforts not only validate the structural principles recently discovered for A. fumigatus cell walls in different morphological stages, but also open up the possibility of extending the current investigation to other fungal materials and cellular systems that are challenging to label.

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.