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Cryo-Electron Tomographic Structure of an Immunodeficiency Virus Envelope Complex In Situ  [PDF]
Giulia Zanetti,John A. G Briggs,Kay Grünewald,Quentin J Sattentau ,Stephen D Fuller
PLOS Pathogens , 2006, DOI: 10.1371/journal.ppat.0020083
Abstract: The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41–gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike.
Cryo-EM Structure of Isomeric Molluscan Hemocyanin Triggered by Viral Infection  [PDF]
Hongtao Zhu, Jun Zhuang, Hongli Feng, Rongfeng Liang, Jiangyong Wang, Lianhui Xie, Ping Zhu
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0098766
Abstract: Hemocyanins (Hcs) of arthropods and mollusks function not only as oxygen transporters, but also as phenoloxidases (POs). In invertebrates, PO is an important component in the innate immune cascade, where it functions as the initiator of melanin synthesis, a pigment involved in encapsulating and killing of pathogenic microbes. Although structures of Hc from several species of invertebrates have been reported, the structural basis for how PO activity is triggered by structural changes of Hc in vivo remains poorly understood. Here, we report a 6.8 ? cryo-electron microscopy (cryo-EM) structure of the isomeric form of hemocyanin, which was isolated from Abalone Shriveling syndrome-associated Virus (AbSV) infected abalone (Halitotis diversicolor), and build a pseudoatomic model of isomeric H. diversicolor hemocyanin 1 (HdH1). Our results show that, compared with native form of HdH1, the architecture of isomeric HdH1 turns into a more relaxed form. The interactions between certain functional units (FUs) present in the native form of Hc either decreased or were totally abolished in the isomeric form of Hc. As a result of that, native state Hc switches to its isomeric form, enabling it to play its role in innate immune responses against invading pathogens.
Cryo-EM Structure of a Novel Calicivirus, Tulane Virus  [PDF]
Guimei Yu, Dongsheng Zhang, Fei Guo, Ming Tan, Xi Jiang, Wen Jiang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0059817
Abstract: Tulane virus (TV) is a newly isolated cultivatable calicivirus that infects juvenile rhesus macaques. Here we report a 6.3 ? resolution cryo-electron microscopy structure of the TV virion. The TV virion is about 400 ? in diameter and consists of a T = 3 icosahedral protein capsid enclosing the RNA genome. 180 copies of the major capsid protein VP1 (~57 KDa) are organized into two types of dimers A/B and C/C and form a thin, smooth shell studded with 90 dimeric protrusions. The overall capsid organization and the capsid protein fold of TV closely resemble that of other caliciviruses, especially of human Norwalk virus, the prototype human norovirus. These close structural similarities support TV as an attractive surrogate for the non-cultivatable human noroviruses. The most distinctive feature of TV is that its C/C dimers are in a highly flexible conformation with significantly reduced interactions between the shell (S) domain and the protruding (P) domain of VP1. A comparative structural analysis indicated that the P domains of TV C/C dimers were much more flexible than those of other caliciviruses. These observations, combined with previous studies on other caliciviruses, led us to hypothesize that the enhanced flexibility of C/C dimer P domains are likely required for efficient calicivirus-host cell interactions and the consequent uncoating and genome release. Residues in the S-P1 hinge between the S and P domain may play a critical role in the flexibility of P domains of C/C dimers.
STUDIES ON STRUCTURE OF CRYO-PVA-HYDROGEL
低温聚乙烯醇(PVA)水凝胶结构的初步研究

HUANG Guang-lin,FENG Yu-ding,HE Jian-ye,
黄光琳
,冯雨丁,贺建业

高分子学报 , 1992,
Abstract: Cryo-hydrogel-rubbery elastomer, containing above 85% water was prepared by freezing aqueous solution of poly (vinyl alcohol) (PVA). Structure of cryo-PVA-hydrogel was studied by TEM. Experimental results showed that the cryo-hydrogel was a non-covalent bonded gel formed due to linking of polymer "clusters".Electron microscopy demonstrates the heterogeneous porous structure of the cryogel. It was found that the porous structure become more and more perfect with increasing the freezing time and polymer concentration. It is considered that phase separation plays an important role in gelation. The freezing is to encourage the phase separation and reinforce the mechanical strength.The results of DSC are in accordance with the results above.
IPET:测定独个生物大分子三维空间结构的实验方法  [PDF]
张腾,彭云辉,童慧敏,Matthew,J,Rames,张磊,任罡
化学进展 , 2013, DOI: 10.7536/PC121105
Abstract: 蛋白质的动态特性和结构活性对于蛋白质功能的调控具有根本意义。传统的结构确定方法(包括X射线和电子显微镜单颗粒分析技术等)往往需要成千上万不同蛋白质分子的平均信号,因此难以确定蛋白质分子的动态结构。而电子显微断层成像技术是一种对独个生物个体结构从不同的观测角度照相、并计算来恢复该个体的三维结构密度图的方法。传统的冷冻电子断层成像重构方法采用整个大尺寸电镜图像进行重构计算,通常用来研究细菌、细胞切片等大尺寸生物个体在较低分辨率下的结构;由于分辨率的限制,不足以获得小尺寸的蛋白质分子的结构细节。最近,任罡研究小组提出一种独个生物颗粒的电子显微断层成像方法(individual-particleelectrontomography,IPET)。该方法通过减小图像尺寸(直至所选区域只包含单个蛋白质分子)的策略,运用提出的FETR(focusedelectrontomographyreconstruction)算法来提高独个大分子重构的分辨率。此方法不需要初始模型和大量分子的平均信号,同时能够容忍一定的测角误差。本文综述了IPET/FETR方法在确定独个分子结构过程中的具体步骤以及如何应用该方法来研究蛋白动态特性和结构变化特征。期望通过该综述和国内同行交流,分享最新的前沿研究,为赶超世界科技前沿的建设添砖加瓦。
Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus  [PDF]
Marek Cyrklaff, Alexandros Linaroudis, Marius Boicu, Petr Chlanda, Wolfgang Baumeister, Gareth Griffiths, Jacomine Krijnse-Locker
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0000420
Abstract: At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction.
Morphology of Influenza B/Lee/40 Determined by Cryo-Electron Microscopy  [PDF]
Garrett Katz, Younes Benkarroum, Hui Wei, William J. Rice, Doris Bucher, Alexandra Alimova, Al Katz, Joanna Klukowska, Gabor T. Herman, Paul Gottlieb
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0088288
Abstract: Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (~83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ~460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.
Limiting factors in single particle cryo electron tomography  [cached]
Mikhail Kudryashev,Daniel Casta?o-Díez,Henning Stahlberg
Computational and Structural Biotechnology Journal , 2012,
Abstract: Modern methods of cryo electron microscopy and tomography allow visualization of protein nanomachines in their native state at the nanometer scale. Image processing methods including sub-volume averaging applied to repeating macromolecular elements within tomograms allow exploring their structures within the native context of the cell, avoiding the need for protein isolation and purification. Today, many different data acquisition protocols and software solutions are available to researchers to determine average structures of macromolecular complexes and potentially to classify structural intermediates. Here, we list the density maps reported in the literature, and analyze each structure for the chosen instrumental settings, sample conditions, main processing steps, and obtained resolution. We present conclusions that identify factors currently limiting the resolution gained by this approach.
Structure Sorting of Multiple Macromolecular States in Heterogeneous Cryo-EM Samples by 3D Multivariate Statistical Analysis  [PDF]
Bruno P. Klaholz
Open Journal of Statistics (OJS) , 2015, DOI: 10.4236/ojs.2015.57081
Abstract:

Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conformational variability of complexes and affects most 3D structure determination methods that rely on signal averaging. Here, an approach is described that allows sorting structural states based on a 3D statistical approach, the 3D sampling and classification (3D-SC) of 3D structures derived from single particles imaged by cryo electron microscopy (cryo-EM). The method is based on jackknifing & bootstrapping of 3D sub-ensembles and 3D multivariate statistical analysis followed by 3D classification. The robustness of the statistical sorting procedure is corroborated using model data from an RNA polymerase structure and experimental data from a ribosome complex. It allows resolving multiple states within heterogeneous complexes that thus become amendable for a structural analysis despite of their highly flexible nature. The method has important implications for high-resolution structural studies and allows describing structure ensembles to provide insights into the dynamics of multi-component macromolecular assemblies.

The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy
Li Xie,QinFen Zhang,XingMeng Lu,XinHong Dai,KunPeng Li,Jian Hong,XuePing Zhou
Science China Life Sciences , 2009, DOI: 10.1007/s11427-009-0151-z
Abstract: Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 . The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 and a single shell thickness of 15 . The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The “Rossmann canyon” is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.
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