Three-dimensional structure of Bax-mediated pores in membrane bilayers

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ∼3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.     

洋葱细胞核中类胀泡结构的数目,体积和分布

在电镜下观察了洋葱（Ａｌｌｉｕｍ ｃｅｐａ Ｌ．）根端分生组织细胞核的连续切片并进行了三维重组。发现：１． 类胀泡结构在细胞核中的数目与二倍染色体数有较大差异。在所观察的Ｓ期细胞核中,该结构数为４—１０ 个;在所观察的Ｇ２ 期细胞核中,该结构数为４—９ 个。２． 类胀泡结构的体积变化比较大,大多数的体积在０．１—０．４ μｍ ３ 之间。不同的Ｓ期细胞核中,类胀泡结构总体积较大者是总体积较小者的１．８ 倍;不同的Ｇ２ 期细胞核中,类胀泡结构总体积较大者是总体积较小者的２．１ 倍。３． 类胀泡结构有集中分布在细胞核内一定区域的倾向。讨论了类胀泡结构是否为着丝粒等问题     

电子显微三维重构技术发展与前沿

本文对电子显微三维重构技术(也称电镜三维重构,electron microscopy 3D reconstruction)进行简要介绍,并在此基础上对该技术当前研究的发展和前沿进行综述,包括高分辨率电镜三维重构、仪器设备性能突破、自动化数据收集和处理、高性能计算技术应用、二/三维图像处理技术的发展和创新、基于三维重构图的模型计算等方面,最后对电子显微三维重构技术的未来进行了展望。     

Cryo-electron microscopy for structural analysis of dynamic biological macromolecules

Background

Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.

Enhancing the contrast of ApoB to locate the surface components in the 3D density map of human LDL

A 26 Å resolution map of the structure of human low-density lipoprotein (LDL) was obtained from electron cryomicroscopy and single-particle image reconstruction. The structure showed a discoidal-shaped LDL particle with high-density regions mainly distributed at the edge of the particle and low-density regions at the flat surface that covers the core region. To determine the chemical components that correspond to these density regions and to delineate the distribution of protein and phospholipid located at the particle surface at the resolution of the map, we used Mono-Sulfo-NHS-Undecagold labeling to increase preferentially the contrast of the apolipoprotein B component on the LDL particle. In the three-dimensional map from the image reconstruction of the undecagold-labeled LDL particles, the high-density region from the undecagold label was distributed mainly at the edge of the particle, and lower density regions were found at the flat surfaces that cover the neutral lipid core. This suggests that apolipoprotein B mainly encircles LDL at the edge of the particle and the phospholipid monolayers are located at the flat surfaces, which are parallel to the cholesterol ester layers in the core and may interact with the core lipid layers through the acyl chains.     

Visualizing a Vibrio cholerae O1 El Tor typing bacteriophage belonging to the Myoviridae group and the packaging of its genomic ends inside the phage capsid

Phage D10, an O1 El Tor tying vibriophage, has been successfully employed to tract the outspread of cholera epidemic. Using Transmission Electron Microscopy and computational image analysis, we have determined the structures of the capsid, head-to-tail connector, the contractile helical tail, the baseplate and combined them to form the complete three-dimensional (3D) D10 phage structure. Using partial denaturation experiments on the genome and using the computed 3D structure of the phage, we have established the packing of the genome ends inside the capsid together with the release styles during the phage infection, respectively. Finally, using the 3D density maps of the different components of the D10 phage, we have presented a simplified picture of morphogenesis of the D10 vibriophage. Using the complete assembled structure of the D10 phage, we have traced the path of the phage genome during the infection process, all the way from the phage head down the tail tube of the tail to the top of the baseplate. To the best of our knowledge, this is first structural study for a long-tailed vibriophage. We have tabulated the structural features of the different components of the phages belonging to the Myoviridae and Siphoviridae. The comparative study suggested the possibility of a common origin of the bacteriophages, irrespective of belonging to different groups and species.     

Hepatitis C virus budding at lipid droplet-associated ER membrane visualized by 3D electron microscopy

The mechanisms underlying hepatitis C virus (HCV) morphogenesis remain elusive, but lipid droplets have recently been shown to be important organelles for virus production. We investigated the interaction between HCV-like particles and lipid droplets by three-dimensional reconstructions of serial ultrathin electron microscopy sections of cells producing the HCV core protein. The budding of HCV-like particles was mostly initiated at membranes close to the lipid droplets rather than at membranes directly apposed to the lipid droplets. This may have important implications for our understanding of the complex relationship between HCV and lipids and may make easier to dissect out the HCV life cycle.     

Structural characterization of dimeric murine aminoacylase III

Aminoacylase III (AAIII) plays an important role in deacetylation of acetylated amino acids and N-acetylated S-cysteine conjugates of halogenated alkenes and alkanes. AAIII, recently cloned from mouse kidney and partially characterized, is a mixture of tetramers and dimers. In the present work, AAIII dimers were purified and shown to be enzymatically active. Limited trypsinolysis showed two domains of approximately 9 and 25 kDa. The three-dimensional structure of the dimer was studied by electron microscopy of negative stained samples and by single-particle reconstruction. A 16A resolution model of the AAIII dimer was created. It has an unusual, cage-like, structure. A realistic AAIII tetramer model was built from two dimers.     

Toward a high-resolution structure of IP3R channel

The ability of cells to maintain low levels of Ca²⁺ under resting conditions and to create rapid and transient increases in Ca²⁺ upon stimulation is a fundamental property of cellular Ca²⁺ signaling mechanism. An increase of cytosolic Ca²⁺ level in response to diverse stimuli is largely accounted for by the inositol 1,4,5-trisphosphate receptor (IP₃R) present in the endoplasmic reticulum membranes of virtually all eukaryotic cells. Extensive information is currently available on the function of IP₃Rs and their interaction with modulators. Very little, however, is known about their molecular architecture and therefore most critical issues surrounding gating of IP₃R channels are still ambiguous, including the central question of how opening of the IP₃R pore is initiated by IP₃ and Ca²⁺. Membrane proteins such as IP₃R channels have proven to be exceptionally difficult targets for structural analysis due to their large size, their location in the membrane environment, and their dynamic nature. To date, a 3D structure of complete IP₃R channel is determined by single-particle cryo-EM at intermediate resolution, and the best crystal structures of IP₃R are limited to a soluble portion of the cytoplasmic region representing ∼15% of the entire channel protein. Together these efforts provide the important structural information for this class of ion channels and serve as the basis for further studies aiming at understanding of the IP₃R function.     

A 3D Graphical Representation of RNA Secondary Structures

Abstract

In this paper, we proposed a 3-D graphical representation of RNA secondary structures. Based on this representation, we outline an approach by constructing a 3-component vector whose components are the normalized leading eigenvalues of the L/L matrices associated with RNA secondary structure. The examination of similarities/dissimilarities among the secondary structure at the 3′-terminus of different viruses illustrates the utility of the approach.     

RNA2D3D: A program for Generating,Viewing, and Comparing 3-Dimensional Models of RNA

Abstract

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.     

SPRING – An image processing package for single-particle based helical reconstruction from electron cryomicrographs

Helical reconstruction from electron cryomicrographs has become a routine technique for macromolecular structure determination of helical assemblies since the first days of Fourier-based three-dimensional image reconstruction. In the past decade, the single-particle technique has had an important impact on the advancement of helical reconstruction. Here, we present the software package SPRING that combines Fourier based symmetry analysis and real-space helical processing into a single workflow. One of the most time-consuming steps in helical reconstruction is the determination of the initial symmetry parameters. First, we propose a class-based helical reconstruction approach that enables the simultaneous exploration and evaluation of many symmetry combinations at low resolution. Second, multiple symmetry solutions can be further assessed and refined by single-particle based helical reconstruction using the correlation of simulated and experimental power spectra. Finally, the 3D structure can be determined to high resolution. In order to validate the procedure, we use the reference specimen Tobacco Mosaic Virus (TMV). After refinement of the helical symmetry, a total of 50,000 asymmetric units from two micrographs are sufficient to reconstruct a subnanometer 3D structure of TMV at 6.4 Å resolution. Furthermore, we introduce the individual programs of the software and discuss enhancements of the helical reconstruction workflow. Thanks to its user-friendly interface and documentation, SPRING can be utilized by the novice as well as the expert user. In addition to the study of well-ordered helical structures, the development of a streamlined workflow for single-particle based helical reconstruction opens new possibilities to analyze specimens that are heterogeneous in symmetries.     

Conical tomography II: A method for the study of cellular organelles in thin sections

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of approximately 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of approximately 4 nm.