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1.
The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, 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 protein. 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. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, 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” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules. 相似文献
2.
Katsuyuki Miyaguchi 《Biology of the cell / under the auspices of the European Cell Biology Organization》2014,106(10):323-345
Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo. 相似文献
3.
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. 相似文献
4.
Thuman-Commike PA 《FEBS letters》2001,505(2):199-205
Three-dimensional structure determination of macromolecules and macromolecular complexes is an integral part of understanding biological functions. For large protein and macromolecular complexes structure determination is often performed using electron cryomicroscopy where projection images of individual macromolecular complexes are combined to produce a three-dimensional reconstruction. Single particle methods have been devised to perform this structure determination for macromolecular complexes with little or no underlying symmetry. These computational methods generally involve an iterative process of aligning unique views of the macromolecular images followed by determination of the angular components that define those views. In this review, this structure determination process is described with the aim of clarifying a seemingly complex structural method. 相似文献
5.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2018,1866(9):973-981
Electron cryotomography (cryo-ET) is an imaging technique uniquely suited to the study of bacterial ultrastructure and cell biology. Recent years have seen a surge in structural and cell biology research on bacteria using cryo-ET. This research has driven major technical developments in the field, with applications emerging to address a wide range of biological questions. In this review, we explore the diversity of cryo-ET approaches used for structural and cellular microbiology, with a focus on in situ localization and structure determination of macromolecules. The first section describes strategies employed to locate target macromolecules within large cellular volumes. Next, we explore methods to study thick specimens by sample thinning. Finally, we review examples of macromolecular structure determination in a cellular context using cryo-ET. The examples outlined serve as powerful demonstrations of how the cellular location, structure, and function of any bacterial macromolecule of interest can be investigated using cryo-ET. 相似文献
6.
Two new species of Parnassia, Parnassia zhengyuana M.X. Ren & J. Zhang and P. simianshanensis M.X. Ren, J. Zhang & Z.Y. Liu, have been found in mountainous areas of Chongqing City, southwest China, and are described and illustrated here. Parnassia zhengyuana is found in a limestone cave near Mt Jinfo, Nanchuan District, Chongqing City. This tiny plant has special spoon‐shaped leaves and undivided staminodes with green apices. Parnassia simianshanensis was discovered in Mt Simian, Jiangjin County, Chongqing City. This species grows along streams on red sandstones, i.e. in Danxia landform. It can be easily identified by its unique rhombic leaves and the five‐ or four‐branched staminodes. The conservation status of P. zhengyuana and P. simianshanensis are assessed respectively as ‘Critically Endangered’ (CR) and ‘VU’ (Vulnerable) according to IUCN criteria. The roles of karst and Danxia landforms in the speciation process are discussed. 相似文献
7.
This article presents a new procedure for the immobilization of macromolecules on gold surfaces, with the purpose of studying macromolecular interactions by simple optical configurations rendering surface plasmon resonance. Gold surfaces were covered by a three-layer structure composed of poly-L-lysine irreversibly bound to gold, followed by a second layer of heparin and a third layer of polylysine. The three-layer structure of polylysine-heparin-polylysine remains irreversibly bound to gold, it prevents biomolecules from coming into direct contact with the metal surface, and it allows the irreversible binding of different proteins and polynucleotides. After binding of a macromolecule to the three-layer structure, the interaction with a second macromolecule can be studied, and then the complex formed by the two interacting macromolecules, together with the second heparin layer and the third polylysine layer, can be broken down just by treatment with an alkaline solution having a pH value above the pK value of the amino groups of polylysine. The first polylysine layer remains irreversibly bound to gold, ready to form a new three-layer structure and, therefore, to support a new macromolecular interaction on the same regenerated surface. Polynucleotide interactions, the proteolytic action of chymotrypsin, and the interaction between the component subunits of a heterotetrameric enzyme are described as examples of macromolecular interactions studied by using this system. The method may be especially suitable for developing of low-cost systems aimed to look for surface resonance signals, and it offers the advantage of allowing calculation of parameters related to the size and stoichiometry of the interacting macromolecules, in addition to the kinetic and equilibrium properties of the interaction. 相似文献
8.
Hancock R 《Biology of the cell / under the auspices of the European Cell Biology Organization》2004,96(8):595-601
Many and possibly all macromolecules in the nucleus are segregated into discrete compartments, but the current model that this is achieved by a fibrillar nuclear matrix which structures the nuclear interior and compartments is not consistent with all experimental observations, as reviewed here. New results are presented which suggest that macromolecular crowding forces play a crucial role in the assembly of at least two compartments, nucleoli and PML bodies, and an in vitro system in which crowding assembles macromolecular complexes into structures which resemble nuclear compartments is described. Crowding forces, which are strong in the nucleus due to the high macromolecule concentration (in the range of 100 mg/ml), vastly increase the association constants of intermolecular interactions and can segregate different macromolecules into discrete phases. The model that they play a role in compartmentalisation of the nucleus is generally consistent with the properties of compartments, including their spherical or quasispherical form and their dynamic and mobile nature. 相似文献
9.
Specimens of the velvety shore bugs (Hemiptera: Ochtheridae) occur in the Early Cretaceous Yixian Formation in north‐eastern China. We describe two new genera and three new species –Pristinochterus ovatus Yao, Zhang & Ren sp. nov., Floricaudus multilocellus Yao, Ren & Shih gen.n. et sp.n. and Angulochterus quatrimaculatus Yao, Zhang & Ren gen.n. et sp.n. – from this deposit. A cladistic analysis based on a combination of fossil and extant taxa, and their morphological characters, clarifies the phylogenetic status of the new fossils and allows the reconstruction of relationships within the family Ochteridae. Two main clades within Ochteridae are recognized from the cladistic analysis: Pristinochterus Yao Cai & Ren and Floricaudus Yao, Ren & Shih gen.n. form a monophyletic lineage; and Angulochterus Yao, Zhang & Ren gen.n. emerges as a sister group to all extant velvety shore bugs. A key to all fossil and extant genera of Ochteridae is provided. 相似文献
10.
11.
A new subfamily Rudisiricinae Gao, Rasnitsyn, Ren & Shih, n. subfam. and a new genus Rudisiricius Gao, Rasnitsyn, Ren & Shih, n. gen. with three new species R. belli Gao, Rasnitsyn, Ren & Shih n. sp., R. crassinodus Gao, Rasnitsyn, Ren & Shih, n. sp., and R. celsus Gao, Rasnitsyn, Ren & Shih, n. sp. are described and illustrated from the family Praesiricidae. The type specimens were collected from the Late Jurassic to Early Cretaceous Yixian Formation, northeastern China. The new subfamily also includes Aulidontes Rasnitsyn from the Upper Jurassic of Karatau in Kazakhstan. This is the first record of Praesiricidae in China. These well-preserved nearly-complete new fossils reported here provide additional material and structure characters about this family, which helps filling some gap in the evolution of Lower Hymenoptera. 相似文献
12.
《Journal of Asia》2021,24(3):866-881
Eight new species of the genus Laena Dejean, 1821 (subfamily Lagriinae, tribe Laenini) from Yunnan Province of China were described and illustrated: L. acutidentata Wei & Ren, sp. nov., L. brevicarina Wei & Ren, sp. nov., L. dongchuana Zhao & Ren, sp. nov., L. glabridentipa Wei & Ren, sp. nov., L. nuda Zhao & Ren, sp. nov., L. raropuncta Wei & Ren, sp. nov., L. rugulosa Wei & Ren, sp. nov. and L. spinicla Wei & Ren, sp. nov.. Illustrations and a key to the known Laena species from Yunnan Province are provided.www.zoobank.org/urn:urn:lsid:zoobank.org:pub:FDEDA1E8-2F2E-4732-8DB4-FE2D7BDC75D8. 相似文献
13.
Wider G 《BioTechniques》2000,29(6):1278-82, 1284-90, 1292 passim
A detailed understanding of the function of a biological macromolecule requires knowledge of its three-dimensional structure. Most atomic-resolution structures of biological macromolecules have been solved either by X-ray diffraction in single crystals or by nuclear magnetic resonance (NMR) in solution. This review surveys the method of NMR structure determination. First, a brief introduction to NMR and its basic concepts is presented. The main part of the article deals with the individual steps necessary for an NMR structure determination. At the end, the discussion turns to considerations on the influence of the molecular size of the macromolecules on the structure determination by NMR. New techniques are discussed that greatly enhance the possibilities of applying NMR to large molecular systems. 相似文献
14.
Tokuriki N Kinjo M Negi S Hoshino M Goto Y Urabe I Yomo T 《Protein science : a publication of the Protein Society》2004,13(1):125-133
Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed. 相似文献
15.
Kerr effect of charged dipolar macromolecules without condensed counterions in conducting solution.
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The theoretical treatment of the Kerr constant of rigid, dipolar, conducting ellipsoidal macromolecules of O'Konski and Krause (1970. J. Phys. Chem. 74:3243) has been extended to very low ionic strength solutions for charged macromolecules. The O'Konski and Krause theoretical treatment postulated a surface conductivity directly on the surface of each macromolecule. For charged macromolecules, this surface conductivity was generally assumed to be caused by movement of condensed counterions on the macromolecules. In the present work, it has been assumed that, at very low ionic strength, the average counterion is at the Debye characteristic distance from the surface of each charged macromolecule and contributes to surface conductivity at that distance, with no additional surface conductivity on the true surface of the macromolecule. Essentially, these considerations change the calculated interaction energy of the macromolecule with an externally applied electric field via a change in both the internal field components and in the reaction field of the macromolecular dipole. The new interaction energy is used to calculate the orientation distribution function of the macromolecules in solution and this distribution function can, in principle, be used to calculate the steady state electric linear or circular dichroism, electric light scattering, anisotropy of conductivity, etc., using the appropriate theoretical treatment for each of these quantities. 相似文献
16.
Lasiosynidae n. fam. is proposed for the genera Lasiosyne Tan, Ren & Chih 2007 (transferred from Archostemata to Polyphaga), Anacapitis Yan 2009 Tarsomegamerus Zhang 2005 (proposed in the superfamily Chrysomeloidea) and Bupredactyla n. gen. The new family is regarded in composition of the infraorder Elateriformia without a more detailed attribution, because it demonstrates a mixture of characters of different families and superfamilies, i.e. somehow an intermediate position between the superfamilies Dascilloidea, Elateroidea, Buprestoidea and Byrrhoidea with most resemblance to Dascillidae, Schizopodidae, Eulichadidae, Ptilodactylidae and Callirrhipidae and probable more close relationship to the last three families. Four new fossil species of the genus Lasiosyne: L. daohugouensis n. sp., L. fedorenkoi n. sp., L. gratiosa n. sp., L. quadricollis n. sp., and also Bupredactyla magna n. sp. are described from the Middle Jurassic Jiulongshan Formation of eastern Inner Mongolia, China. A probable generic composition of the new family is considered. The synonymy of generic names Anacapitis Yan 2009 and Brachysyne Tan & Ren 2009, n. syn. as well as synonymy of species names Lasiosyne euglyphea Tan, Ren & Chih 2007, Pappisyne eucallus Tan & Ren 2009, n. syn. and Pappisyne spathulata Tan & Ren 2009, n. syn. are proposed. 相似文献
17.
David M. Belnap Abhinav Kumar Jon T. Folk Thomas J. Smith Timothy S. Baker 《Journal of structural biology》1999,125(2-3)
Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15–40 Å), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction. 相似文献
18.
Since its debut in the mid 1970s, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single-layered or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes. 相似文献
19.
三维电子显微镜方法进展 总被引:3,自引:0,他引:3
从生物样品(细胞或大分子)的电镜图象重组其三维结构的方法近年取得了重大进展,这是冷冻电镜样品制备、电镜设备、图象处理和分析方法等几方面进步的综合结果。三维电镜方法的进步和完善,使细胞和学家得以了解在复杂的细胞过程中各种相关细胞器之间的空间关系,而使分子生物学家不仅可以研究那些能够形成二维结晶的样品,并为分析具有重要生物功能但不能形成二维结晶的大分子或分子聚休物的结构提供了一种强大的手段。 相似文献
20.
Trinh MH Odorico M Pique ME Teulon JM Roberts VA Ten Eyck LF Getzoff ED Parot P Chen SW Pellequer JL 《Structure (London, England : 1993)》2012,20(1):113-120
Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures. 相似文献