Reoviridae ; Cryoelectron Microscopy

Humans ; Dynorphins/metabolism* ; Receptors, Opioid, kappa/metabolism* ; Cryoelectron Microscopy

Cryoelectron Microscopy ; methods ; Crystallography, X-Ray ; Image Processing, Computer-Assisted ; methods ; Models, Molecular ; Molecular Conformation

Animals ; Chromatin ; genetics ; ultrastructure ; Cryoelectron Microscopy ; Epigenesis, Genetic ; Humans ; Models, Molecular ; Molecular Conformation

G protein-coupled receptors(GPCRs)represent the largest class of cell surface receptors,mediating wide range of cellular and physiological processes through their transducers,G proteins and the-arrestins participate in almost all pathological processes. Recent technological advances are revolutionizing the utility of cryo-electron microscopy(cryo-EM),leading to a tremendous progress in the structural studies of biological macromolecules and cryo-EM has played a leading role in the structural biology of GPCR signaling complex. New discoveries of high-resolution threedimensional structures of GPCR signaling complexes based on cryo-EM have emerged vigorously,which depict the common structural characteristics of intermolecular interaction between GPCR and G protein complex-the conformational changes of the transmembrane helix 6 of receptors,and also demonstrate the structural basis of G protein subtype selectivity. Single-particle cryo-EM becomes an efficient tool for identifying the molecular mechanism of receptor-ligand interaction,providing important information for understanding GPCR signaling and the structure-based drug design.
Cryoelectron Microscopy ; Protein Binding ; Protein Structure, Tertiary ; Receptors, G-Protein-Coupled ; chemistry

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc²155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.
Anti-Bacterial Agents/pharmacology* ; Cryoelectron Microscopy ; Mycobacterium/genetics* ; Ribosomal Proteins/genetics* ; Ribosomes/metabolism*

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.
Animals ; Artificial Intelligence ; Cryoelectron Microscopy ; Nuclear Pore/ultrastructure* ; Oocytes/metabolism* ; Xenopus laevis

Cryo-electron microscopy (cryo-EM) imaging has the unique potential to bridge the gap between cellular and molecular biology. Therefore, cryo-EM three-dimensional (3D) reconstruction has been rapidly developed in recent several years and applied widely in life science research to reveal the structures of large macromolecular assemblies and cellular complexes, which is critical to understanding their functions at all scales. Although the technical breakthrough in recent years, for example, the introduction of the direct detection device (DDD) camera and the development of cryo-EM software tools, made the three cryo-EM pioneers share the 2017 Nobel Prize, several bottleneck problems still exist that hamper the further increase of the resolution of single-particle reconstruction and hold back the application of in situ subnanometer structure determination by cryo-tomography. Radiation damage is still the key limiting factor in cryo-EM. In order to minimize the radiation damage and preserve as much resolution as possible, the imaging conditions of a low dose and weak contrast make cryo-EM images extremely noisy with very low signal-to-noise ratios (SNR), generally about 0.1. The high noise will obscure the fine details in cryo-EM images or reconstructed maps. Thus, a method to reduce the level of noise and improve the resolution has become an important issue. In this paper, we systematically reviewed and compared some robust filters in the cryo-EM field of two aspects, single-particle analysis (SPA) and cryo-electron tomography (cryo-ET), and especially studied their applications, such as, 3D reconstruction, visualization, structural analysis, and interpretation. Conventional approaches to noise reduction in cryo-EM imaging include the use of Gaussian, median, and bilateral filters, among other means. A Gaussian filter selects an appropriate filter kernel to conduct spatial convolution with a noisy image. Although noise with larger standard deviations in cryo-EM images can be suppressed and satisfactory performance is achieved in certain cases, this filter also blurs the images and over-smooths small-scale image features. This is especially detrimental when precise quantitative information needs to be extracted. Unlike a Gaussian filter, a median filter is based on the order statistics of the image and selects the median intensity in a window of the adjacent pixels to denoise the image. Although this filter is robust to outliers, it suffers from aliasing problems that possibly result in incorrect information for cryo-EM structure interpretation. A bilateral filter is a nonlinear filter that performs spatial weighted averaging and is more selective in the pixels allowing to contribute to the weighted sum, excluding the high frequency noise from the smoothing process. Thus, this filter can be used to smooth out noise while maintaining the edge details, which is similar to an anisotropic diffusion filter, and distinct from a Gaussian filter but its utility will be limited when the SNR of a cryo-EM image is very low. Generally, spatial filtering methods have the disadvantage of losing image resolution when reducing noise. A wavelet transform can exploit the wavelet's natural ability to separate a signal from noise at multiple image scales to allow for joint resolution in both the spatial and frequency domains, and thus has the potential to outperform existing methods. The modified wavelet shrinkage filter we developed can offer a remarkable improvement in image quality with a good compromise between detail preservation and noise smoothing. We expect that our review study on different filters can provide benefits to cryo-EM applications and the interpretation of biological structures.
Algorithms ; Cryoelectron Microscopy ; Image Processing, Computer-Assisted ; Normal Distribution ; Signal-To-Noise Ratio ; Tomography, X-Ray Computed

The adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg2+ shows a "head-to-tail" dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.
Siderophores/metabolism* ; Iron/metabolism* ; Mycobacterium tuberculosis/metabolism* ; Cryoelectron Microscopy ; Adenosine Triphosphate/metabolism* ; ATP-Binding Cassette Transporters

Heme/metabolism* ; Cryoelectron Microscopy ; Membrane Transport Proteins ; Escherichia coli Proteins/metabolism*