Characterization of a full-length infectious clone of bovine foamy virus 3026

The biological features of most foamy viruses(FVs) are poorly understood, including bovine foamy virus(BFV). BFV strain 3026(BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid(AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.

Orienting the future of bio-macromolecular electron microscopy

With 40 years of development, bio-macromolecule cryo-electron microscopy（cryo-EM） has completed its revolution in terms of resolution and currently plays a highly important role in structural biology study. According to different specimen states, cryo-EM involves three specific techniques： single-particle analysis（SPA）, electron tomography and subtomogram averaging, and electron diffraction. None of these three techniques have realized their full potential for solving the structures of bio-macromolecules and therefore need additional development. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which include the air–water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimens, and electron radiation damage. Furthermore, potential solutions of these bottlenecks worthy of further investigation are proposed and discussed.

A local-optimization refinement algorithm in single particle analysis for macromolecular complex with multiple rigid modules

Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macro- molecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate align- ments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orien- tations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimen- sional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large （60S/50S） and the small （40S/30S） subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolutionsingle particle analysis of macromolecular complexes with dynamic conformations.

AutoGDeterm： Automatic Geometry Determination for Electron Tomography

Electron Tomography （ET） is an important method for studying cell ultrastructure in three-dimensional （3D） space. By combining cryo-electron tomography of frozen-hydrated samples （cryo-ET） and a sub-tomogram averaging approach, ET has recently reached sub-nanometer resolution, thereby realizing the capability for gaining direct insights into function and mechanism. To obtain a high-resolution 3D ET reconstruction, alignment and geometry determination of the ET tilt series are necessary. However, typical methods for determining geometry require human intervention, which is not only subjective and easily introduces errors, but is also labor intensive for high-throughput tomographic reconstructions. To overcome these problems, we have developed an automatic geometry-determination method, called AutoGDeterm. By taking advantage of the high-contrast re-projections of the Iterative Compressed-sensing Optimized Non-Uniform Fast Fourier Transform （NUFFT） reconstruction （ICON） and a series of numerical analysis methods, AutoGDeterm achieves high-precision fully automated geometry determination. Experimental results on simulated and resin-embedded datasets show that the accuracy of AutoGDeterm is high and comparable to that of the typical ＂manual positioning＂ method. We have made AutoGDeterm available as software, which can be freely downloaded from our website http：//ear.ict.ac.cn.

秀丽隐杆线虫RNA结合蛋白复合物AMG-1/SLRP-1通过线粒体稳态维持实现调控生殖腺发育和精子发生

RNA结合蛋白(mtRBP)介导的mRNA转录后调节对精子发生必不可少但却鲜有报道.在本文中,我们鉴定到一个在生殖腺中特异性表达的线粒体RNA结合蛋白AMG-1,它是秀丽隐杆线虫精子发生过程中必需的蛋白,同时与哺乳动物LRPPRC蛋白同源.amg-1突变会阻碍生殖腺的发育,最终导致生殖细胞的线粒体形态和结构异常以及线粒体功能障碍.通过测序鉴定RNA结合蛋白的靶点发现,AMG-1更倾向于与mtDNA编码的参与线粒体核糖体组装的12S和16S核糖体RNA(rRNA)结合,12S rRNA对于维持生殖细胞线粒体蛋白稳态至关重要,而12S rRNA的表达却受AMG-1蛋白调节.此外,哺乳动物SLIRP在秀丽线虫中的同源蛋白SLRP-1蛋白与AMG-1在遗传上存在互作关系,它们可共同调节秀丽线虫的精子发生和育性.综上所述,这些发现揭示了mtRBP蛋白AMG-1在线粒体调控中的新机制,这可能为由线粒体功能障碍引发的男性不育治疗提供新的理论基础.

Three-dimensional reconstruction of Picea wilsonii Mast. pollen grains using automated electron microscopy

Three-dimensional electron microscopy(3 D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3 D reconstruction of biological samples remains challenging. Herein, we present a 3 D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and38.57 nm lateral resolution using AutoCUTS-SEM(automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P.wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.

Structural characterization of coatomer in its cytosolic state

Studies on coat protein I （COPI） have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single- particle analyses, and also performing CXMS （chemical cross-linking coupled with mass spectrometry） for vali- dation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

8 Å structure of the outer rings of the Xenopus laevis nuclear pore complex obtained by cryo-EM and AI

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 8Åresolution 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 4.9Å.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.

Supramolecular assembly of chloroplast NADH dehydrogenase-like complex with photosystem Ⅰfrom Arabidopsis thaliana

Cyclic electron transport/flow(CET/CEF)in chloroplasts is a regulatory process essential for the optimization of plant photosynthetic efficiency.A crucial CEF pathway is catalyzed by a membrane-embedded NADH dehydrogenase-like(NDH)complex that contains at least 29 protein subunits and associates with photosystem I(PSI)to form the NDH-PSI supercomplex.Here,we report the 3.9Åresolution structure of the Arabidopsis thaliana NDH-PSI(AtNDH-PSI)supercomplex.We constructed structural models for 26 AtNDH subunits,among which 11 are unique to chloroplasts and stabilize the core part of the NDH complex.In the supercomplex,one NDH can bind up to two PSI-light-harvesting complex I(PSI-LHCI)complexes at both sides of its membrane arm.Two minor LHCIs,Lhca5 and Lhca6,each present in one PSI-LHCI,interact with NDH and contribute to supercomplex formation and stabilization.Collectively,our study reveals the structural details of the AtNDH-PSI supercomplex assembly and provides a molecular basis for further investigation of the regulatory mechanism of CEF in plants.

Three-dimensional visualization of arsenic stimulated mouse liver sinusoidal by FIB-SEM approach

Dear Editor,Liver sinusoidal endothelial cells （LSECs） are the most abundant non-parenchymal and highly specialized fenes- trated cells in the liver. LSECs differ from endothelial cells of other capillaries due to the presence of open fenestrations and the absence of a basal lamina （Wisse et al., 1996; Braet and Wisse, 2002; Aird, 2007; Cogger et at., 2008）. During the differentiation process, LSECs become fenestrated to facilitate the transfer of circulating nutrients, lipids, and lipoproteins between blood and the space of Disse for normal liver metabolism. Only particles smaller than the fenestrae can reach the hepatocytes or leave the space of Disse （Wisse et al., 1985）. The size of fenestration changes upon different conditions to regulate the substrate exchange.

细胞器的发生:核糖体作为组织者和实施者

When viewed as a broad concept,organelle biogenesis is considered the main event of cytoplasm formation,during which the vesicle precursors of cellular organelles are translocated and biosynthesized with biomolecular inputs and assembly.In this paper,we sum up the recent research advances on the phenomenon known as“organelle biogenesis.”