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In JoVE (1)
- क्रायो - इलेक्ट्रॉन टोमोग्राफी और स्वचालित उप - tomogram औसत का उपयोग एचआईवी लिफाफा glycoproteins की आणविक संरचना के निर्धारण
Other Publications (12)
- Journal of Structural Biology
- Journal of Structural Biology
- Journal of Structural Biology
- The Journal of Biological Chemistry
- Journal of Bacteriology
- Journal of Structural Biology
- PLoS Pathogens
- The EMBO Journal
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Virology
- Journal of Structural Biology
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Articles by Mario J. Borgnia in JoVE
क्रायो - इलेक्ट्रॉन टोमोग्राफी और स्वचालित उप - tomogram औसत का उपयोग एचआईवी लिफाफा glycoproteins की आणविक संरचना के निर्धारण
Joel R. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology
प्रोटोकॉल के एक उच्च throughput झिल्ली प्रोटीन का उपयोग क्रायो - इलेक्ट्रॉन टोमोग्राफी और 3 डी इमेज प्रोसेसिंग के ढांचे का निर्धारण दृष्टिकोण का वर्णन करता है. यह नमूना तैयारी के विवरण, डाटा संग्रह, डाटा प्रोसेसिंग और व्याख्या को शामिल किया गया है, और दृष्टिकोण, एचआईवी -1 लिफाफा ग्लाइकोप्रोटीन के लिए एक प्रतिनिधि लक्ष्य के उत्पादन के साथ समाप्त. इन कम्प्यूटेशनल प्रक्रियाओं के लिए एक तरीका है कि छात्रों और शोधकर्ताओं के लिए सक्षम बनाता दूर से काम करने के लिए और डाटा प्रोसेसिंग और संरचनात्मक विश्लेषण करने के लिए योगदान में डिजाइन किए हैं.
Other articles by Mario J. Borgnia on PubMed
A Core-weighted Fitting Method for Docking Atomic Structures into Low-resolution Maps: Application to Cryo-electron Microscopy
Journal of Structural Biology. Jan, 2003 | Pubmed ID: 12576021
Cryo-electron microscopy of "single particles" is a powerful method to analyze structures of large macromolecular assemblies that are not amenable to investigation by traditional X-ray crystallographic methods. A key step in these studies is to obtain atomic interpretations of multiprotein complexes by fitting atomic structures of individual components into maps obtained from electron microscopic data. Here, we report the use of a "core-weighting" method, combined with a grid-threading Monte Carlo (GTMC) approach for this purpose. The "core" of an individual structure is defined to represent the part where the density distribution is least likely to be altered by other components that comprise the macromolecular assembly of interest. The performance of the method has been evaluated by its ability to determine the correct fit of (i) the alpha-chain of the T-cell receptor variable domain into a simulated map of the alphabeta complex at resolutions between 5 and 40 A, and (ii) the E2 catalytic domain of the pyruvate dehydrogenase into an experimentally determined map, at 14 A resolution, of the icosahedral complex formed by 60 copies of this enzyme. Using the X-ray structures of the two test cases as references, we demonstrate that, in contrast to more traditional methods, the combination of the core-weighting method and the grid-threading Monte Carlo approach can identify the correct fit reliably and rapidly from the low-resolution maps that are typical of structures determined with the use of single-particle electron microscopy.
Automated Image Acquisition and Processing Using a New Generation of 4K X 4K CCD Cameras for Cryo Electron Microscopic Studies of Macromolecular Assemblies
Journal of Structural Biology. Aug, 2003 | Pubmed ID: 12972350
We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope. In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4 K CCD cameras for use in cryo electron microscopic applications. We demonstrate that at 120 kV, and at a nominal magnification of 67000 x, power spectra and signal-to-noise ratios for the new 4 K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as approximately 1/6.5A(-1). The specimen area imaged for each exposure on the 4 K CCD is about one-third of the area that can be recorded with a similar exposure on film. The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole. The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 A for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.
Visualization of Alpha-helical Features in a Density Map Constructed Using 9 Molecular Images of the 1.8 MDa Icosahedral Core of Pyruvate Dehydrogenase
Journal of Structural Biology. Aug, 2004 | Pubmed ID: 15193642
Strategies to achieve the highest resolutions in structures of protein complexes determined by cryo-electron microscopy generally involve averaging information from large numbers of individual molecular images. However, significant limitations are posed by heterogeneity in image quality and in protein conformation that are inherent to large data sets of images. Here, we demonstrate that the combination of iterative refinement and stringent molecular sorting is an effective method to obtain substantial improvements in map quality of the 1.8 MDa icosahedral catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus. From a starting set of 42,945 images of the core complex, we show that using only the best 139 particles in the data set produces a map that is superior to those constructed with greater numbers of images, and that the location of many of the alpha-helices in the structure can be unambiguously visualized in a map constructed from as few as 9 particles.
Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy
The Journal of Biological Chemistry. Feb, 2006 | Pubmed ID: 16308322
The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.
Three-dimensional Imaging of the Highly Bent Architecture of Bdellovibrio Bacteriovorus by Using Cryo-electron Tomography
Journal of Bacteriology. Apr, 2008 | Pubmed ID: 18203829
Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.
Journal of Structural Biology. Oct, 2008 | Pubmed ID: 18585059
Tomograms of biological specimens derived using transmission electron microscopy can be intrinsically noisy due to the use of low electron doses, the presence of a "missing wedge" in most data collection schemes, and inaccuracies arising during 3D volume reconstruction. Before tomograms can be interpreted reliably, for example, by 3D segmentation, it is essential that the data be suitably denoised using procedures that can be individually optimized for specific data sets. Here, we implement a systematic procedure to compare various nonlinear denoising techniques on tomograms recorded at room temperature and at cryogenic temperatures, and establish quantitative criteria to select a denoising approach that is most relevant for a given tomogram. We demonstrate that using an appropriate denoising algorithm facilitates robust segmentation of tomograms of HIV-infected macrophages and Bdellovibrio bacteria obtained from specimens at room and cryogenic temperatures, respectively. We validate this strategy of automated segmentation of optimally denoised tomograms by comparing its performance with manual extraction of key features from the same tomograms.
Nature. Sep, 2008 | Pubmed ID: 18668044
The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.
Molecular Architectures of Trimeric SIV and HIV-1 Envelope Glycoproteins on Intact Viruses: Strain-dependent Variation in Quaternary Structure
PLoS Pathogens. 2010 | Pubmed ID: 21203482
The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ~20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively "open" conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.
Lateral Density of Receptor Arrays in the Membrane Plane Influences Sensitivity of the E. Coli Chemotaxis Response
The EMBO Journal. May, 2011 | Pubmed ID: 21441899
In chemotactic bacteria, transmembrane chemoreceptors, CheA and CheW form the core signalling complex of the chemotaxis sensory apparatus. These complexes are organized in extended arrays in the cytoplasmic membrane that allow bacteria to respond to changes in concentration of extracellular ligands via a cooperative, allosteric response that leads to substantial amplification of the signal induced by ligand binding. Here, we have combined cryo-electron tomographic studies of the 3D spatial architecture of chemoreceptor arrays in intact E. coli cells with computational modelling to develop a predictive model for the cooperativity and sensitivity of the chemotaxis response. The predictions were tested experimentally using fluorescence resonance energy transfer (FRET) microscopy. Our results demonstrate that changes in lateral packing densities of the partially ordered, spatially extended chemoreceptor arrays can modulate the bacterial chemotaxis response, and that information about the molecular organization of the arrays derived by cryo-electron tomography of intact cells can be translated into testable, predictive computational models of the chemotaxis response.
Trimeric HIV-1 Glycoprotein Gp140 Immunogens and Native HIV-1 Envelope Glycoproteins Display the Same Closed and Open Quaternary Molecular Architectures
Proceedings of the National Academy of Sciences of the United States of America. Jul, 2011 | Pubmed ID: 21709254
The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.
Three-dimensional Structures of Soluble CD4-bound States of Trimeric Simian Immunodeficiency Virus Envelope Glycoproteins Determined by Using Cryo-electron Tomography
Journal of Virology. Dec, 2011 | Pubmed ID: 21937655
The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.
Computational Separation of Conformational Heterogeneity Using Cryo-electron Tomography and 3D Sub-volume Averaging
Journal of Structural Biology. Jan, 2012 | Pubmed ID: 22248450
We have previously used cryo-electron tomography combined with sub-volume averaging and classification to obtain 3D structures of macromolecular assemblies in cases where a single dominant species was present, and applied these methods to the analysis of a variety of trimeric HIV-1 and SIV envelope glycoproteins (Env). Here, we extend these studies by demonstrating automated, iterative, missing wedge-corrected 3D image alignment and classification methods to distinguish multiple conformations that are present simultaneously. We present a method for measuring the spatial distribution of the vector elements representing distinct conformational states of Env. We identify data processing strategies that allow clear separation of the previously characterized closed and open conformations, as well as unliganded and antibody-liganded states of Env when they are present in mixtures. We show that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual Env sub-volumes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity in heterogeneous mixtures. We validate these procedures for computational separation by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded as well as open and closed conformations of Env present simultaneously in mixtures.