块染技术在生物样品超微结构三维重构中的应用

该文主要探究生物大分子和各种细胞器的空间位置、相互作用的细节信息,对解析生命过程至关重要。因此,通过体电子显微学技术,实现大尺度生物样品的超微结构的三维重构,对促进细胞生物学、神经生物学等的研究具有重要意义。然而,生物样品本身只能提供微弱的电子反差,电镜成像后样品的细节结构不清晰。染色技术可以有效地增大样品的电子散射差异,提高样品超微结构的电镜图像质量。近年来,已有大量研究使用块染技术实现了大尺度样品的超微结构成像,该文通过概述电镜样品的制备过程、染色方法和染色原理,比较了在块染过程中不同的桥联剂和块染剂的特点,以期为促进块染技术的应用和发展提供有效思路。     

结构生物学研究方法的重大突破——电子直接探测相机在冷冻电镜中的应用

近年来,科学家应用冷冻电镜技术(cryo-EM)解析出了低对称性生物大分子的高分辨率(3~5魡)三维结构,并用其密度图直接进行了分子建模。与传统的X-射线和NMR方法相比,冷冻电镜技术具有适用于分子量较大的生物分子、样品不需结晶且用量很少等优势。尤其是电子直接探测相机(electron direct detection device,DDD)在冷冻电镜技术中的应用,使高分辨率的结构研究变得更加简单、应用更为广泛,是一个重大突破。文章介绍DDD相机的原理和技术优势,及其在解决冷冻电镜技术困难中的一些应用,进而展望了DDD相机可能给冷冻电镜技术应用带来的突破性进展。     

二维核磁共振与核酸的溶液结构

二维核磁共振技术(2D-NMR)是近十几年出现的核磁共振方法的一个分支,已能在溶液态测定蛋白质、核酸等生物大分子的三维结构,成为与单晶X-射线研究相互补充的重要手段.与蛋白质的二维核磁共振研究相比,核酸的2D-NMR研究起步略晚,但近三、五年来已取得长足的进步,在作核酸的结构测定、研究核酸—蛋白质相互作用中发挥了其     

超低温含水生物样品电子显微镜技术

现代电子显微镜的分辨力已达到0.2nm,但用它观察生物结构,其分辨力几乎要低一个数量级。目前电镜观察生物结构的最好记录是对紫膜结构的研究,能显示1nm的规则结构,表明紫膜具有二维结晶性质。对无规则生物结构的观察,以核糖体为例,其分辨力只及3—5nm。因此,目前生物样品电子显微镜分析     

遗传信息传递的晶体学解密

生物大分子(如蛋白质等)在生物体内行使功能必须要保证其立体结构的正确性。作为研究大分子高级结构的主要手段,结晶技术结合X-射线衍射技术、核磁共振技术以及电镜技术被普遍应用于高级结构的数据分析。随着这些技术的进一步完善,目前已经能完成蛋白质与配体的共结晶。遗传信息从最原始的DNA或RNA传递到以蛋白质的形式呈现功能的过程是由众多酶或蛋白质复合体催化的多步骤进程,解析其中重要元件的空间结构推动了对这些酶反应机理的深入研究。主要阐述结晶技术在遗传信息传递过程中关键酶或蛋白质复合体的研究中的应用。     

生物高分辨电子显微学进展

生物高分辨电子显微学是近年来发展起来的一种可与X射线晶体学相媲美的测定生物大分子高分辨结构的方法．它克服了一些限制X射线晶体学应用的困难,可以直接对非晶体状态的生物大分子或仅能形成二维晶体的蛋白进行结构测定．这一技术主要包括高分辨电子显微象的获得与电子显微象解析．文章就这一技术应用中的一些问题：自然结构的保持、辐射损伤、低衬度、低信噪比等进行了讨论．     

综述: 冷冻电镜技术在表观遗传学研究中的应用

染色质装配、修饰和重塑复合体,以及它们和核小体、染色质等一起形成的超大分子复合体的精细结构解析,对于在原子水平揭示表观遗传信息建立、维持和调控的分子机制至关重要．近年来,迅速发展的冷冻电镜三维重构技术对于解析这些多亚基、大分子质量、柔性超大分子复合体的结构带来了很好的机遇．本文综述了冷冻电镜三维重构技术在表观遗传学相关的结构研究领域中的一些应用和进展．     

冷冻电镜技术在表观遗传学研究中的应用

染色质装配、修饰和重塑复合体,以及它们和核小体、染色质等一起形成的超大分子复合体的精细结构解析,对于在原子水平揭示表观遗传信息建立、维持和调控的分子机制至关重要.近年来,迅速发展的冷冻电镜三维重构技术对于解析这些多亚基、大分子质量、柔性超大分子复合体的结构带来了很好的机遇.本文综述了冷冻电镜三维重构技术在表观遗传学相关的结构研究领域中的一些应用和进展.     

利用INTERNET进行蛋白质结构的检索、图象显示和模型构建

生物大分子的三维结构是了解生物分子功能的前提,对于分子生物学家、细胞生物学家和生物化学家建立生物过程的分子机制越来越重要。近年来生物大分子的三维结构信息增长极快。据纽约Brookhaven国家实验室Protein Data Bank(PDB)统计的数据,从1992至1995年该库收集的生物大分子结构的数目分别是1007,1727,2921和3821,平均年递增50％。面对如此大量而且不断增加的结构数据,如何快速准确     

电子断层成像技术及其在细胞生物学中的应用

电子断层成像技术（electrontomography）是近年来发展起来一项三维成像技术,可以在纳米分辨率（2-10nm）水平上获得生物大分子及其复合物或聚集体、细胞器、细胞以及组织的三维结构,而且可以用于研究生物大分子在细胞中的定位、排列、分布以及相互作用,已逐渐成为细胞生物学领域中的一项重要技术手段。该文针对这项技术及其在细胞生物学中的应用作一简要介绍。     

A system for the three-dimensional construction,manipulation and display of microbiological models

A system is described for building up serial sections into a three dimensional structure, incorporating density, that can be displayed and then further manipulated by rotation about three orthogonal axes. The initial application was to produce a computer model of a protein structure and to compare the diverse images obtained from rotation with the two dimensional images observed in related electron micrographs. To obtain sufficient contrast in the electron microscope images of protein structures, the specimens need to be stained and since this can cause some deformation of the observed images, it is also necessary to simulate the possible effects of stain on the protein model. Because of the need to compare numerous orientations of the combined model, techniques are available either for speeding up the comparison or for obtaining better accuracy. The methods have been applied to the interpretation of electron micrograph images of microbiological specimens, where the three dimensional structure of the specimen is an important aid in understanding its biological function, but the techniques are also applicable to more general serial reconstruction requirements.     

核基质附着区广泛参与基因组三维结构折叠的生物信息学分析

在细胞分裂间期,每条染色质都占据着特定的染色质领域（chromosome territory,CT）。每个CT领域内进一步分成不同的拓扑学相关区域（topological associated domain,TAD）,每个TAD又由若干子TAD（sub-TAD）构成。不同的TAD相互聚集,形成基因活跃表达和不表达的A、B两种组份或区室（compartment）。然而,目前对于染色质折叠方式及维持机制的研究尚无定论。核基质附着区（matrix attachment regions,MARs）是在不同物种基因组中广泛存在的一类富含AT序列的与核基质结合的DNA元件,能够通过与CTCF、SATB1等调控蛋白质相互作用,对远距离的基因表达进行调控。本研究以染色质三维结构为背景,通过整合染色质三维结构及组蛋白修饰等组学数据,对MARs元件与染色质三维结构的关系进行研究,对MARs元件参与形成的相互作用网络的结构及功能进行探索。结果发现,MARs元件与染色质三维结构高度相关,而且在高强度相互作用中占据较大的比例,提示MARs元件在染色质折叠方面发挥作用。此外,通过拓扑结构聚类分析还首次揭示,MARs元件分为不同类型,包括维持染色质领域及空间构象等的结构单元部分,以及调控基因表达等的功能单元部分。这表明,MARs元件在基因组三维高级结构的建立、维持以及功能等方面发挥重要作用。     

What can proteomic analyses contribute to understanding the molecular biology and clinical behavior of prostate cancer?

Identifying the proteins and their complex interactions that promote and/or sustain the aggressive malignant phenotype is essential for understanding key effectors of the molecular biology of prostate cancer. This is also essential for development of new clinical applications. A variety of proteomic techniques, ranging from mass spectrometry to new methods of multiplexing protein identification, have great potential for rapidly achieving these goals. However, in order to obtain meaningful results, these techniques must be applied within the context of our knowledge of the heterogeneity of prostate tissues and tumors, the impact of specimen processing on both the quality and quantity of proteins detected and a thorough understanding of prostate cell biology. Collaboration between the protein chemist and the prostate cell biologist will expedite progress in this important field.     

What can proteomic analyses contribute to understanding the molecular biology and clinical behavior of prostate cancer?

Identifying the proteins and their complex interactions that promote and/or sustain the aggressive malignant phenotype is essential for understanding key effectors of the molecular biology of prostate cancer. This is also essential for development of new clinical applications. A variety of proteomic techniques, ranging from mass spectrometry to new methods of multiplexing protein identification, have great potential for rapidly achieving these goals. However, in order to obtain meaningful results, these techniques must be applied within the context of our knowledge of the heterogeneity of prostate tissues and tumors, the impact of specimen processing on both the quality and quantity of proteins detected and a thorough understanding of prostate cell biology. Collaboration between the protein chemist and the prostate cell biologist will expedite progress in this important field.     

Scanning electron microscopic observations of the ependymal cell and the subependymal layer of the third brain ventricle in the rat

This investigation was undertaken to clarify the three dimensional ultrastructure of the subependymal layer in relation with the ependymal cell layer in rat brain using the scanning electron microscope (SEM). The subependymal layer existing below the ependyma of the third ventricle in the brain of mature albino rats was examined with S E M. The hypothalamus freshly excised after median sagittal section was treated by collagenase with or without trypsin for a short while to remove the ependymal cells at the ventricular wall. After the enzymatic pretreatment of the specimen, many ependymal cells were removed and the subependymal layer was partially exposed. Most of the ciliated ependymal cells remaining at the ventricular wall extended long, single basal processes which then penetrated into the subependymal layer. The subependymal layer was composed of a delicate framework of thin processes of glial cells, ependymal cells and, in addition nerve cells. Scattered among the neuropil just beneath the ependymal cell layer, there were relatively small, globular subependymal cells. Occasionally, there were large bundles of unmyelinated nerve fibres in the subependymal layer. The individual nerve fibres distinctly showed many axonal varicosities within the fibres. Intermingled with the nerve fibres, glial processes of various forms were present. The structure of the ependymal cells and the subependymal layer was compared with the findings already reported in the studies using light and transmission electron microscope.     

108 Individual-particle electron tomography: a method for studying macromolecule dynamics

In solution, macromolecules are naturally flexible and dynamic. Dynamic personalities and structural heterogeneities of macromolecules are essential to understanding their proper function (Karplus & Kuriyan, 2005). However, structural determination of dynamic/heterogeneous macromolecules is limited by current technology such as: X-ray crystallography, nuclear magnetic resonance spectrum, small angle scattering, and electron microscopy single-particle reconstruction. A common weakness of all current techniques is requiring an averaged signal from thousands to millions of different macromolecules. Using averaged “signal” must involve in an assumption that macromolecules remain in identical structures or few identical conformations. This assumption is a good estimate for some macromolecule that have a rigid body, but not for most macromolecules that have “soft”, flexible, and dynamic body, such as lipoproteins and antibodies. An ideal approach for structure determination regardless of macromolecular dynamics is to use non-averaged signal, i.e. the signal from a single macromolecule itself. We developed a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance macromolecule (Zhang & Ren, 2012). FETR can tolerate certain levels of image distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. Since this approach can obtain the structure of a single-instance macromolecule, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ‘‘snapshot’’ of molecule structure determination (Zhang & Ren, 2012). FETR/IPET provides a completely new opportunity for a single-macromolecule structure determination, and could be used to study the dynamic character, equilibrium fluctuation, to reveal macromolecular mechanism, and even to track the intermediate state of the reaction of macromolecules (Zhang et al., 2010; Zhang & Ren, 2010).     

Tilted specimen in the electron microscope: A simple specimen holder and the calculation of tilt angles for crystalline specimens

A simple specimen holder is described for a Siemens electron microscope which will allow the specimen grid to be set at inclinations up to 75° to the electron beam in any azimuthal direction. This device is suitable for measuring tilted images for the three-dimensional reconstruction of crystalline specimens. A method is also described for calculating the tilt angles for such crystalline specimens by comparing the unit cell dimensions in tilted and untilted images.     

Comparative modelling of protein structure and its impact on microbial cell factories

Comparative modeling is becoming an increasingly helpful technique in microbial cell factories as the knowledge of the three-dimensional structure of a protein would be an invaluable aid to solve problems on protein production. For this reason, an introduction to comparative modeling is presented, with special emphasis on the basic concepts, opportunities and challenges of protein structure prediction. This review is intended to serve as a guide for the biologist who has no special expertise and who is not involved in the determination of protein structure. Selected applications of comparative modeling in microbial cell factories are outlined, and the role of microbial cell factories in the structural genomics initiative is discussed.     

Cell fine structures observed by scanning electron microscopy]

The scanning electron microscope (SEM) provides vivid seemingly three dimensional images which are easier to understand for us than transmission electron microscopic images. For this point of view scanning electron microscopy is advantageous in morphological researches of cell fine structures. Nevertheless, there were few studies in this field, because SEM had much lower resolution than transmission electron microscope (TEM) and because there was no adequate method to reveal intracellular structures. In recent years, however, the resolution of SEM has been markedly improved and the specimen preparation techniques have also advanced. In this paper, some of our preparation technique for revealing cell surface structures or intracellular structures, in particular, osmium-DMSO-osmium method, and the results observed by these methods were described. 1) Nucleus. The nucleus was wrapped with a nuclear envelope that consisted of two membranes enclosing a narrow space. On the surface of the envelope many nuclear pores were observed. 2) Endoplasmic reticulum (ER). Rough ER consisted of flattened cisternae, arranged in parallel. The surface were studded with many ribosomes which were often arranged spirally to form polysomes. Smooth ER consisted of tubules. 3) Golgi complex. a) The Golgi stacks were all linked by anastomosing. b) Connection between Golgi stacks and rough ER was often observed. c) Cisternae in a Golgi stack were connected each other. 4) Mitochondria. The mitochondrion was bounded by 2 sheets of unit membrane and the inner membrane projected into the interior of the organelles to make mitochondrial cristae.     

Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments

Understanding the molecular mechanism of muscle contraction and its regulation has been greatly influenced and aided by studies of myofilament structure in invertebrate muscles. Invertebrates are easily obtained and cover a broad spectrum of species and functional specializations. The thick (myosin-containing) filaments from some invertebrates are especially stable and simple in structure and thus much more amenable to structural analysis than those of vertebrates. Comparative studies of invertebrate filaments by electron microscopy and image processing have provided important generalizations of muscle molecular structure and function. This article reviews methods for preparing thick and thin filaments from invertebrate muscle, for imaging filaments by electron microscopy, and for determining their three dimensional structure by image processing. It also highlights some of the key insights into filament function that have come from these studies.