使用电子断层技术对IgG1抗体逐个分子的三维重构与结构分析

抗体(antibody)又称免疫球蛋白(immunoglobulin,Ig),是人体免疫反应的重要参与者.了解抗体的结构和结构动态特征,是理解人体免疫作用机理、修复或提高免疫能力、定向设计抗体以治疗各种疾病的基础.本文以人体IgG1抗体为对象,综述了使用透射电子显微学方法研究IgG1抗体结构方向的最新进展.详细介绍了使用逐个分子的电子断层三维重构技术(individual-particle electron tomography,IPET)对抗体进行结构研究的方法,包括样品制备、图像处理和数据分析等.并描述了利用该技术,在研究抗体结合肽分子后的结构形变和通过收集不同构象来研究抗体动态结构特征方面所取得的阶段性成果.最后,对尚待解决的关键问题与该技术未来的发展方向进行了讨论与展望.     

冷冻电子断层成像技术及其在生物研究领域的应用

冷冻电子断层成像可以在纳米级尺度上研究那些结构不具有均一性的分子、病毒、细胞器以及它们之间组成的复合体的三维结构。在过去的十年中,电子显微镜硬件、冷冻制样设备和技术,以及自动化断层数据收集方法的进步使得本研究领域得到快速发展。本文对冷冻电子断层成像的方法,包括基本原理、样品制备、断层数据采集和图像处理、三维重构以及重建信息的理解和展示、近年来在生物样品领域的一些典型应用以及前景作一简单介绍。     

猪动脉内皮细胞胞膜小窝的电子断层三维结构分析

利用电子断层三维重构技术对猪动脉内皮细胞 (porcine aorta endothelial cell,PAE cell) 胞膜小窝的三维结构进行了初步研究,发现胞膜小窝在细胞膜表面呈不均匀分布并在局部形成聚集,胞膜小窝膜内外表面都由宽度约14～16 nm的条纹状结构所环绕,推测该条纹状结构主要由小窝蛋白和胆固醇构成,狭窄的胞膜小窝颈部区域存在高密度的丝状结构．三维结构显示胞膜小窝与纤维丝体网络(推测为微管网络)相互作用,暗示了细胞内吞可能的运输途径．     

Low-Dose Automated Electron Tomography: A Recent Implementation

Low-dose automated tomography has been implemented on a 400-kV JEOL intermediate voltage electron microscope. Instrumentation and procedures for automatic tomographic series data collection are described. Difficulties encountered and ways to overcome them are discussed. A low-dose tomographic projection series of a triad junction of frog sartorius muscle was semiautomatically collected and a 3-D reconstruction of this organelle was made.     

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.     

Gifa V. 4: A complete package for NMR data set processing

Summary The Gifa program is designed for processing, displaying and analysing 1D, 2D and 3D NMR data sets. It has been constructed in a modular fashion, based on three independent modules: a set of commands that perform all the basic processing operations such as apodisation functions, a complete set of Fourier Transforms, phasing and baseline correction, peak-picking and line fitting, linear prediction and maximum entropy processing; a set of command language primitives that permit the execution of complex macro commands; and a set of graphic commands that permit to build a complete graphic user interface, allowing the user to interact easily with the program. We have tried to create a versatile program that can be easily extended according to the user's requirements and that is adapted to a novice as well as an experienced user. The program runs on any UNIX computer, with or without graphic display, in interactive or batch mode.     

ConfocalVR: Immersive Visualization for Confocal Microscopy

ConfocalVR is a virtual reality (VR) application created to improve the ability of researchers to study the complexity of cell architecture. Confocal microscopes take pictures of fluorescently labeled proteins or molecules at different focal planes to create a stack of two-dimensional images throughout the specimen. Current software applications reconstruct the three-dimensional (3D) image and render it as a two-dimensional projection onto a computer screen where users need to rotate the image to expose the full 3D structure. This process is mentally taxing, breaks down if you stop the rotation, and does not take advantage of the eye's full field of view. ConfocalVR exploits consumer-grade VR systems to fully immerse the user in the 3D cellular image. In this virtual environment, the user can (1) adjust image viewing parameters without leaving the virtual space, (2) reach out and grab the image to quickly rotate and scale the image to focus on key features, and (3) interact with other users in a shared virtual space enabling real-time collaborative exploration and discussion. We found that immersive VR technology allows the user to rapidly understand cellular architecture and protein or molecule distribution. We note that it is impossible to understand the value of immersive visualization without experiencing it first hand, so we encourage readers to get access to a VR system, download this software, and evaluate it for yourself. The ConfocalVR software is available for download at http://www.confocalvr.com, and is free for nonprofits.     

Coding nucleic acids are chaperons for protein folding: A novel theory of protein folding

The arguments for nucleic acid chaperons are reviewed and three new lines of evidence are added. (1) It was found that amino acids encoded by codons in short nucleic acid loops frequently form turns and helices in the corresponding protein structures. (2) The amino acids encoded by partially complementary (1st and 3rd nucleotides) codons are more frequently co-located in the encoded proteins than expected by chance. (3) There are significant correlations between thermodynamic changes (ddG) caused by codon mutations in nucleic acids and the thermodynamic changes caused by the corresponding amino acid mutations in the encoded proteins. We conclude that the concept of the Proteomic Code and nucleic acid chaperons seems correct from the bioinformatics point of view, and we expect to see direct biochemical experiments and evidence in the near future.