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.     

冷冻电镜单颗粒三维重构密度掩模的自动生成方法研究

冷冻电镜单颗粒三维重构技术是用来解析生物大分子三维结构的常用方法.然而目前在单颗粒三维重构过程中,溶剂平滑操作还存在一定缺陷:没有一款主流的单颗粒三维重构程序能够自动寻找掩模(mask)三维密度图,使得三维重构过程难免受到噪音统计学模型计算偏差的干扰.为解决这一问题,本研究借鉴X射线晶体学中解析优化相位所广泛采用的溶剂平滑方法,采用高斯滤波、坎尼边缘检测、最小误差阈值处理等方法处理重构所得三维密度图,优化溶剂平滑操作,发展在单颗粒三维重构过程中自动寻找mask三维密度图的方法.运用三维密度图傅里叶壳层相关系数(fourier shell correlation,FSC)曲线图、模拟颗粒数据重构角度误差散点图等指标评估此方法的效果.结果表明,自动寻找mask密度图的方法能够较好地找到涵盖分子结构信号区域的mask密度图,较为明显提高三维重构所得密度图分辨率.     

三维电子显微学自动化数据采集的进展

生物三维电子显微学在过去几年取得了巨大的突破,一些具有高对称性的病毒颗粒获得了准原子分辨率的结构,非对称性的生物大分子及其复合体的结构分辨率也有快速的提高。而要获得高分辨率的结构,获取足够多的高质量电子显微照片是其中的一个关键因素。近年来,自动化数据采集技术在电子断层成像术和单颗粒方法中都取得了很大的进展。其广泛应用将使结构测定更加快速并使结构分辨率提高到更高的层次。     

生物三维电子显微学进入全新高速发展时期

生物三维电子显微学主要由三个部分组成——电子晶体学、单颗粒技术和电子断层成像术,其结构解析对象的尺度范围介于x射线晶体学与光学显微镜之间,适合从蛋白质分子结构到细胞和组织结构的解析。以冷冻电镜技术与三维重构技术为基础的低温电子显微学代表了生物电子显微学的前沿。低温单颗粒技术对于高度对称的病毒颗粒的解析最近已达到3．8A分辨率,正在成为解析分子量很大的蛋白质复合体高分辨结构的有效技术手段。低温电子断层成像技术目前对于真核细胞样品的结构解析已达到约40A的分辨率,在今后5年有望达到20A。这样,把x射线晶体学、NMR以及电镜三维重构获得的蛋白质分子及复合体的高分辨率的结构,锚定到较低分辨率的电子断层成像图像中,从而在细胞水平上获得高精确的蛋白质空间定位和原子分辨率的蛋白质相互作用的结构信息。这将成为把分子水平的结构研究与细胞水平的生命活动衔接起来的可行途径。     

用于蛋白质同源模建及三维结构预测的结构比较方法

继前面的工作把测试蛋白从三族扩大到十一族,寻求联配参数的普适“缺省值“;比较不同的主链曲率和挠率的计算方法,进一步确认主链的折红红分几何刻划方法的有效性;寻找有效的可变缺失突变惩罚函数的形式。结果表明,编制的蛋白质多重联配软件系统是满意的,可用于蛋白质三维结构预测。     

导管三维重构的基本方法

导管是被子植物运输水分和无机盐的管道,它由一串导管分子以穿孔端壁相互衔接而成。关于它在输水过程中的作用,常用的比较方法是通过离析来测定导管分子的长度和直径,或者是通过三切面来观察某一导管分子与邻近细胞的相互关系。由于上述方法所展示的仅仅是某一局部区域中木质部各种组成分子的平面关系或者是某一组成分子的变化模式,所     

白色念珠菌羊毛甾醇14α—去甲基化酶三维结构分子？…

基于四种原核细胞色素Ｐ４５０晶体蛋白Ｐ４５０ＢＭ３、Ｐ４５０ｃａｍ、Ｐ４５０ｔｅｒｐ、４５０ｅｒｙＦ模建白色念珠菌羊毛甾醇１４α－去甲基化酶三维结构。序列匹配采用四种晶体结构比较结果基础之上提出的细胞色素Ｐ４５０超家族蛋白基于结构知识的序列匹配方法。以Ｐ４５０ＢＭ３晶体结构坐标模建目标蛋白结构保守区主链结构,结构保守区侧链构象来源于四种晶体蛋白与模建蛋白对应残基同源性得分最高残基构象。建模结果用分     

CD_(126)功能域三维结构研究进展

ＣＤ１２６功能域三维结构的研究对研制具有不同生物学活性的新型ＩＬ－６Ｒ突变体和进行基于ＣＤ１２６三维结构的药物分子设计研究有重要的指导意义。本文概述ＣＤ１２６空间结构预测及同源模建的研究进展,并总结ＣＤ１２６与其配基ＩＬ－６形成高亲和力受体复合物时构－效关系分析的研究动态 。     

老年性痴呆症患者海马老年斑三维重构

老年斑是老年性痴呆 (AD)的主要病理特征之一。β 淀粉样蛋白 (Aβ)在老年斑中的积聚与AD发病关系密切。因此 ,如何抑制老年斑内Aβ的积聚和促进Aβ纤维的解聚对于治疗和预防AD有十分重要的意义。通过免疫组织化学染色、MATLAB软件图像处理、AVS重构等技术构建了老年斑三维结构。实验发现海马部位的弥散斑和经典斑具有孔状结构 ;弥散斑无明显核心结构 ,密度分布趋势由中心向周围递减 ;经典斑中心Aβ沉积最致密 ,周围Aβ沉积最少 ,而斑块外周Aβ沉积密度居中。老年斑三维结构重构方法的建立 ,有助于老年斑积聚动力学的研究 ,并对AD病理机制的探讨具有重要意义。     

电子技术在医学影像处理中的发展与应用

80年代未至90年代初,随着最新计算机图像处理技术与生物医学工程技术的发展,为医学成像提供了有力的工具,人体组织的三维显现不再以二维投影或横断面序列实现,三维图像真实的再显终于成为可能。本文介绍了国外当前影像技术的最新发展,着重于三维图像的显示、定位、分割及多种技术组合成像,并附上了相当数量的图片展示其在不同医疗领域的应用。     

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.     

3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.     

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.     

用DNA银染法研究传染性软疣病毒的形态发生发育过程

用ＤＮＡ银染技术显示了传染性软疣病毒（ＭＣＶ）形态发生发育及其过程中ＤＮＡ的变化。结果显示：在被感染的皮肤表皮细胞内都有一个大小及构型不同的银染区（病毒工厂）。ＭＣＶ的发生发育均在银染区内而不在胞质区内。其发生过程是先在细胞一侧的胞质内复制合成病毒ＤＮＡ等物质,形成中等密度的银粒大小不等的银染区（病毒前包涵体区）,然后在其中形成致密的细粒状银染区（病毒前基质区）,接着在后者的周围出现弧形的粗粒银沉淀（初期ＭＣＶ的外膜）,逐渐分割包围病毒前基质而形成初期ＭＣＶ。在发育过程中,初期ＭＣＶ的外膜、基质,核心外膜及核心均经过一系列的形态变化。侧体是中期ＭＣＶ向成熟期发育中出现的暂时性结构,其本质为含ＤＮＡ成分的病毒基质。本研究提示：ＭＣＶ的ＤＮＡ物质进入皮肤表皮细胞后能大量复制,合成大量的病毒蛋白质,自主地装配成完整的初期ＭＣＶ,后者有独特的形态发育过程。     

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.     

Novel phantom for performance evaluation of contrast-enhanced 3D rotational angiography

PurposeThis technical note presents an in-house phantom with a specially designed contrast-object module constructed to address the need for three-dimensional rotational angiography (3DRA) testing.MethodsThe initial part of the study was a brief evaluation on the commercially available phantom used for 3DRA and computed tomography angiography (CTA) to confirm the need for a special phantom for 3D angiography. Once confirmed, an in-house phantom was constructed. The novel phantom was tested to evaluate the basic image performance metrics, i.e., unsharpness (MTF) and noise characterization (NPS), as well as to show its capability for vessel contrast visibility study.ResultsThe low contrast objects in the commercially available tools dedicated for CT is found to yield significantly lower signal difference to noise ratio (SDNR) when used for 3DRA, therefore deemed inadequate for 3DRA contrast evaluation. The constructed in-house phantom demonstrates a capability to serve for basic imaging performance check (MTF, NPS, and low contrast evaluation) for 3DRA and CTA. With higher and potentially adjustable visibility of contrast objects as artificial vessels, the in-house phantom also makes more clinically relevant tests, e.g., human- or model observer study and task-based optimization, possible.ConclusionThe novel phantom with special contrast object module shows higher visibility in 3DRA compared to the currently available commercial phantom and, therefore, is recommended for use in 3D angiography.     

3D Coronary Artery Reconstruction by 2D Motion Compensation Based on Mutual Information

Background

3D reconstruction of the coronary arteries can provide more information in the interventional surgery. Motion compensation is one kind of the 3D reconstruction method.

Development and validation of a generic 3D model of the distal femur

The development and validation of a virtual generic 3D model of the distal femur using computer graphical methods is presented. The synthesis of the generic model requires the following steps: acquisition of bony 3D morphology using standard computed tomography (CT) imaging; alignment of 3D models reconstructed from CT images with a common coordinate system; computer graphical sectioning of the models; extraction of bone contours from the image sections; combining and averaging of extracted contours; and 3D reconstruction of the averaged contours.

The generic models reconstructed from the averaged contours of six cadaver femora were validated by comparing their surface geometry on a point to point basis with that of the CT reconstructed reference models. The mean errors ranged from 0.99 to 2.5 mm and were in agreement with the qualitative assessment of the models.     

Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K_a ~ 10⁵), the binding of the intact protein to F-actin is strong (apparent K_a ~ 10⁸), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat–F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.