Journal of structural biology最新文献

Atrial fibrillation (AF) is the most common clinical arrhythmia, however there is limited understanding of its pathophysiology including the cellular and ultrastructural changes rendered by the irregular rhythm, which limits pharmacological therapy development. Prior work has demonstrated the importance of reactive oxygen species (ROS) and mitochondrial dysfunction in the development of AF. Mitochondrial structure, interactions with other organelles such as sarcoplasmic reticulum (SR) and T-tubules (TT), and degradation of dysfunctional mitochondria via mitophagy are important processes to understand ultrastructural changes due to AF. However, most analysis of mitochondrial structure and interactome in AF has been limited to two-dimensional (2D) modalities such as transmission electron microscopy (EM), which does not fully visualize the morphological evolution of the mitochondria during mitophagy. Herein, we utilize focused ion beam-scanning electron microscopy (FIB-SEM) and perform reconstruction of three-dimensional (3D) EM from murine left atrial samples and measure the interactions of mitochondria with SR and TT. We developed a novel 3D quantitative analysis of FIB-SEM in a murine model of AF to quantify mitophagy stage, mitophagosome size in cardiomyocytes, and mitochondrial structural remodeling when compared with control mice. We show that in our murine model of spontaneous and continuous AF due to persistent late sodium current, left atrial cardiomyocytes have heterogenous mitochondria, with a significant number which are enlarged with increased elongation and structural complexity. Mitophagosomes in AF cardiomyocytes are located at Z-lines where they neighbor large, elongated mitochondria. Mitochondria in AF cardiomyocytes show increased organelle interaction, with 5X greater contact area with SR and are 4X as likely to interact with TT when compared to control. We show that mitophagy in AF cardiomyocytes involves 2.5X larger mitophagosomes that carry increased organelle contents. In conclusion, when oxidative stress overcomes compensatory mechanisms, mitophagy in AF faces a challenge of degrading bulky complex mitochondria, which may result in increased SR and TT contacts, perhaps allowing for mitochondrial Ca²⁺ maintenance and antioxidant production.

Parkinson’s disease (PD) is a category of neurodegenerative disorders (ND) that currently lack comprehensive and definitive treatment strategies. The etiology of PD can be attributed to the presence and aggregation of a protein known as α-synuclein. Researchers have observed that the application of an external electrostatic field holds the potential to induce the separation of the fibrous structures into peptides. To comprehend this phenomenon, our investigation involved simulations conducted on the α-synuclein peptides through the application of Molecular Dynamics (MD) simulation techniques under the influence of a 0.1 V/nm electric field. The results obtained from the MD simulations revealed that in the presence of external electric field, the monomer and oligomeric forms of α-synuclein are experienced significant conformational changes which could prevent them from further aggregation. However, as the number of peptide units in the model system increases, forming trimers and tetramers, the stability against the electric field also increases. This enhanced stability in larger aggregates indicates a critical threshold in α-synuclein assembly where the electric field’s effectiveness in disrupting the aggregation diminishes. Therefore, our findings suggest that early diagnosis and intervention could be crucial in preventing PD progression. When α-synuclein predominantly exists in its monomeric or dimeric form, applying even a lower electric field could effectively disrupt the initial aggregation process. Inhibition of α-synuclein fibril formation at early stages might serve as a viable solution to combat PD by halting the formation of more stable and pathogenic α-synuclein fibrils.

Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a ∼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a ∼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine’s capability for high-resolution data acquisition and screening on lower-end systems by obtaining ∼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM.

Atomic force microscope enables ultra-precision imaging of living cells. However, atomic force microscope imaging is a complex and time-consuming process. The obtained images of living cells usually have low resolution and are easily influenced by noise leading to unsatisfactory imaging quality, obstructing the research and analysis based on cell images. Herein, an adaptive attention image reconstruction network based on residual encoder-decoder was proposed, through the combination of deep learning technology and atomic force microscope imaging supporting high-quality cell image acquisition. Compared with other learning-based methods, the proposed network showed higher peak signal-to-noise ratio, higher structural similarity and better image reconstruction performances. In addition, the cell images reconstructed by each method were used for cell recognition, and the cell images reconstructed by the proposed network had the highest cell recognition rate. The proposed network has brought insights into the atomic force microscope-based imaging of living cells and cell image reconstruction, which is of great significance in biological and medical research.

Osteosarcoma (OS) is the most common malignant primary bone tumor in humans and occurs in various subtypes. Tumor formation happens through malignant osteoblasts producing immature bone. In the present paper we studied two different subtypes of osteosarcoma, from one individual with conventional OS with massive sclerosis and one individual with parosteal OS, based on a multimodal approach including small angle x-ray scattering (SAXS), wide angle x-ray diffraction (WAXS), backscattered electron imaging (BEI) and Raman spectroscopy. It was found that both tumors showed reduced mineral particle sizes and degree of orientation of the collagen-mineral composite in the affected areas, alongside with a decreased crystallinity. Distinct differences between the tumor material from the two individuals were found in the degree of mineralization. Further differences were observed in the carbonate to phosphate ratio, which is related to the degree of carbonate substitution in bone mineral and indicative of the turnover rate. The contraction of the c-axis of the bone mineral crystals proved to be a further, very sensitive parameter, potentially indicative of malignancy.

Human serum albumin (HSA) is the most prevalent plasma protein in the human body, accounting for 60 % of the total plasma protein. HSA plays a major pharmacokinetic function, serving as a facilitator in the distribution of endobiotics and xenobiotics within the organism. In this paper we report the cryoEM structures of HSA in the apo form and in complex with two ligands (salicylic acid and teniposide) at a resolution of 3.5, 3.7 and 3.4 Å, respectively. We expand upon previously published work and further demonstrate that sub-4 Å maps of ∼60 kDa proteins can be routinely obtained using a 200 kV microscope, employing standard workflows. Most importantly, these maps allowed for the identification of small molecule ligands, emphasizing the practical applicability of this methodology and providing a starting point for subsequent computational modeling and in silico optimization.

Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call “honeycomb gold discs”, replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.

Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

This study synthesized and evaluated a series of benzotriazole derivatives denoted 3(a-j) and 6(a-j) for their anti-HIV-1 RT activities compared to the standard drug efavirenz. Notably, compound 3 h, followed closely by 6 h, exhibited significant anti-HIV-1 RT efficacy relative to the standard drug. In vivo oral toxicity studies were conducted for the most active compound 3 h, confirming its nontoxic nature to ascertain the safety profile. By employing molecular docking techniques, we explored the potential interactions between the synthesized compounds (ligands) and a target biomolecule (protein)(PDB ID 1RT2) at the molecular level. We undertook the molecular dynamics study of 3 h, the most active compound, within the active binding pocket of the cocrystallized structure of HIV-1 RT (PDB ID 1RT2). We aimed to learn more about how biomolecular systems behave, interact, and change at the atomic or molecular level over time. Finally, the DFT-derived HOMO and LUMO orbitals, as well as analysis of the molecular electrostatic potential map, aid in discerning the reactivity characteristics of our molecule.