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Andrew B Ward - One of the best experts on this subject based on the ideXlab platform.
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model building and refinement of a natively glycosylated hiv 1 env protein by high resolution Cryoelectron Microscopy
Structure, 2015Co-Authors: Dmitry Lyumkis, Andrew B WardAbstract:Summary Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle Cryoelectron Microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 A resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.
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model building and refinement of a natively glycosylated hiv 1 env protein by high resolution Cryoelectron Microscopy
Structure, 2015Co-Authors: Dmitry Lyumkis, Andrew B Ward, Jeong Hyun Lee, Natalia De ValAbstract:Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle Cryoelectron Microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 A resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.
Timothy S. Baker - One of the best experts on this subject based on the ideXlab platform.
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antibody mediated neutralization of human rhinovirus 14 explored by means of Cryoelectron Microscopy and x ray crystallography of virus fab complexes
Journal of Virology, 1998Co-Authors: Zhiwei Che, Timothy S. Baker, Norman H Olson, Donna Leippe, Wai Ming Lee, Anne G Mosser, Roland R Rueckert, Thomas J SmithAbstract:The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and Cryoelectron Microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the beta-B-beta-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.
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comparison of the native ccmv virion with in vitro assembled ccmv virions by Cryoelectron Microscopy and image reconstruction
Virology, 1998Co-Authors: James M Fox, Guoji Wang, Timothy S. Baker, John E. Johnson, Jeffrey A Speir, N H Olson, Mark J YoungAbstract:Cryoelectron Microscopy and three-dimensional image reconstruction analysis has been used to determine the structure of native andin vitroassembled cowpea chlorotic mottle virus (CCMV) virions and capsids to 25-A resolution. Purified CCMV coat protein was used in conjunction within vitrotranscribed viral RNAs to assemble RNA 1 only, RNA 2 only, RNA 3/4 only, and empty (RNA lacking) virions. The image reconstructions demonstrate that thein vitroassembled CCMV virions are morphologically indistinguishable from native virions purified from infected plants. The viral RNA (vRNA) is packaged similarly within the different types of virions. The centers of all assembled particles are generally devoid of density and the vRNA packs against the interior surface of the virion shell. The vRNA appears to adopt an ordered conformation at each of the quasi-threefold axes.
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The structure of cucumber mosaic virus: Cryoelectron Microscopy, X-ray crystallography, and sequence analysis.
Virology, 1997Co-Authors: William R. Wikoff, Chao Jo Tsai, Guoji Wang, Timothy S. Baker, John E. JohnsonAbstract:The three-dimensional structure of cucumber mosaic virus (CMV) was analyzed at 23 A u resolution by Cryoelectron Microscopy and image reconstruction, demonstrating structural similarity to cowpea chlorotic mottle virus (CCMV), another member of the Bromoviridae family. The CMV structure was determined at 8 A u resolution by X-ray crystallography with phases determined by single isomorphous replacement and refined by fivefold noncrystallographic symmetry averaging. The X-ray structure agreed with the electron Microscopy reconstruction; the electron density is consistent with b-barrel subunits arranged with TA 3 quasi-symmetry in an orientation similar to that observed in CCMV. Strong density surrounding the icosahedral threefold axes (quasi sixfold axes in the T A 3 particle) between 80 and 100 A u from the particle center formed a cylinder of radius 11 A u , similar to the density observed in the same region of CCMV. This density corresponds to the b-annulus of CCMV, which differentiates hexamers from pentamers and determines the formation of the T A 3 particles. The CMV and CCMV amino acid sequences were aligned, providing information (based on the CCMV atomic model) about the probable distribution of residues in the three-dimensional structure of CMV. q 1997 Academic Press
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IRIS Explorer Software for Radial-Depth Cueing Reovirus Particles and Other Macromolecular Structures Determined by Cryoelectron Microscopy and Image Reconstruction
Journal of structural biology, 1997Co-Authors: Stephan Spencer, Timothy S. Baker, Jean-yves Sgro, Kelly A. Dryden, Max L NibertAbstract:Abstract Structures of biological macromolecules determined by transmission Cryoelectron Microscopy (cryo-TEM) and three-dimensional image reconstruction are often displayed as surface-shaded representations with depth cueing along the viewed direction (Z cueing) Depth cueing to indicate distance from the center of virus particles (radial-depth cueing, or R cueing) has also been used. We have found that a style of R cueing in which color is applied in smooth or discontinuous gradients using the IRIS Explorer software is an informative technique for displaying the structures of virus particles solved by cryo-TEM and image reconstruction. To develop and test these methods, we used existing cryo-TEM reconstructions of mammalian reovirus particles. The newly applied visualization techniques allowed us to discern several new structural features, including sites in the inner capsid through which the viral mRNAs may be extruded after they are synthesized by the reovirus transcriptase complexes. To demonstrate the broad utility of the methods, we also applied them to cryo-TEM reconstructions of human rhinovirus, native and swollen forms of cowpea chlorotic mottle virus, truncated core of pyruvate dehydrogenase complex from Saccharomyces cerevisiae, and flagellar filament of Salmonella typhimurium. We conclude that R cueing with color gradients is a useful tool for displaying virus particles and other macromolecules analyzed by cryo-TEM and image reconstruction.
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a model based approach for determining orientations of biological macromolecules imaged by Cryoelectron Microscopy
Journal of Structural Biology, 1996Co-Authors: Timothy S. Baker, Holland R ChengAbstract:A polar Fourier transform (PFT) method is described that facilitates determination and refinement of orientations of individual biological macromolecules imaged with Cryoelectron Microscopy techniques. A three-dimensional density map serves as a high signal-to-noise model from which a PFT database of different views is generated and against which the PFTs of individual images are correlated. The PFT produces rotation-invariant data particularly well-suited for rapid and accurate determination of orientation parameters. The method relies on accurate knowledge of the center of symmetry and radial scale of both model and image data but is insensitive to the relative contrast and background values of these data. Density maps may be derived from a variety of sources such as computer-generated models, X-ray crystallographic structures, and three-dimensional reconstructions computed from images. The PFT technique has been particularly useful for the analysis of particles with icosahedral symmetry and could be adapted for the analysis of single particles of any symmetry for which a crude model exists or can be produced.
Jeong Hyun Lee - One of the best experts on this subject based on the ideXlab platform.
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model building and refinement of a natively glycosylated hiv 1 env protein by high resolution Cryoelectron Microscopy
Structure, 2015Co-Authors: Dmitry Lyumkis, Andrew B Ward, Jeong Hyun Lee, Natalia De ValAbstract:Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle Cryoelectron Microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 A resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.
Phoebe L. Stewart - One of the best experts on this subject based on the ideXlab platform.
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Cryoelectron Microscopy analysis of small heat shock protein 16 5 hsp16 5 complexes with t4 lysozyme reveals the structural basis of multimode binding
Journal of Biological Chemistry, 2013Co-Authors: Jian Shi, Phoebe L. Stewart, Hanane A Koteiche, Ezelle T Mcdonald, Tara Fox, Hassane S MchaourabAbstract:Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used Cryoelectron Microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.
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Cryoelectron Microscopy and epr analysis of engineered symmetric and polydisperse hsp16 5 assemblies reveals determinants of polydispersity and substrate binding
Journal of Biological Chemistry, 2006Co-Authors: Jian Shi, Hanane A Koteiche, Hassane S Mchaourab, Phoebe L. StewartAbstract:We have identified sequence and structural determinants of oligomer size, symmetry, and polydispersity in the small heat shock protein super family. Using an insertion mutagenesis strategy that mimics evolutionary sequence divergence, we induced the ordered oligomer of Methanococcus jannaschii Hsp16.5 to transition to either expanded symmetric or polydisperse assemblies. A hybrid approach combining spin labeling EPR and Cryoelectron Microscopy imaging at 10A resolution reveals that the underlying plasticity is mediated by a packing interface with minimal contacts and a flexible C-terminal tether between dimers. Twenty-four dimeric building blocks related by octahedral symmetry assemble into the expanded symmetric oligomer. In contrast, the polydisperse variant has an ordered dimeric building block that heterogeneously packs to yield oligomers of various sizes. Increased exposure of the N-terminal region in the Hsp16.5 variants correlates with enhanced binding to destabilized mutants of T4 lysozyme, whereas deletion of this region reduces binding. Transition to larger intermediates with enhanced substrate binding capacity has been observed in other small heat shock proteins including lens α-crystallin mutants linked to congenital cataract. Together, these results provide a mechanistic perspective on substrate recognition and binding by the small heat shock protein superfamily.
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visualization of α helices in a 6 angstrom resolution Cryoelectron Microscopy structure of adenovirus allows refinement of capsid protein assignments
Journal of Virology, 2006Co-Authors: Susan D Saban, Mariena Silvestry, Glen R Nemerow, Phoebe L. StewartAbstract:The structure of adenovirus was determined to a resolution of 6 A by Cryoelectron Microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that α-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed α-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly α-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in ∼1.5 copies per hexon trimer and is predicted to have two long α-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long α-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long α-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal α-helix from protein IX.
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Cryoelectron Microscopy imaging of recombinant and tissue derived vaults localization of the mvp n termini and vparp
Journal of Molecular Biology, 2004Co-Authors: Yeshi Mikyas, Miriam Makabi, Sujna Ravalfernandes, Lea Harrington, Phoebe L. Stewart, Valerie A. Kickhoefer, Leonard H. RomeAbstract:The vault is a highly conserved ribonucleoprotein particle found in all higher eukaryotes. It has a barrel-shaped structure and is composed of the major vault protein (MVP); vault poly(ADP-ribose) polymerase (VPARP); telomerase-associated protein 1 (TEP1); and small untranslated RNA (vRNA). Although its strong conservation and high abundance indicate an important cellular role, the function of the vault is unknown. In humans, vaults have been implicated in multidrug resistance during chemotherapy. Recently, assembly of recombinant vaults has been established in insect cells expressing only MVP. Here, we demonstrate that co-expression of MVP with one or both of the other two vault proteins results in their co-assembly into regularly shaped vaults. Particles assembled from MVP with N-terminal peptide tags of various length are compared. Cryoelectron Microscopy (cryoEM) and single-particle image reconstruction methods were used to determine the structure of nine recombinant vaults of various composition, as well as wild-type and TEP1-deficient mouse vaults. Recombinant vaults with MVP N-terminal peptide tags showed internal density that varied in size with the length of the tag. Reconstruction of a recombinant vault with a cysteine-rich tag revealed 48-fold rotational symmetry for the vault. A model is proposed for the organization of MVP within the vault with all of the MVP N termini interacting non-covalently at the vault midsection and 48 copies of MVP forming each half vault. CryoEM difference mapping localized VPARP to three density bands lining the inner surface of the vault. Difference maps designed to localize TEP1 showed only weak density inside of the caps, suggesting that TEP1 may interact with MVP via a small interaction region. In the absence of atomic-resolution structures for either VPARP or TEP1, fold recognition methods were applied. A total of 21 repeats were predicted for the TEP1 WD-repeat domain, suggesting an unusually large β-propeller fold.
Dmitry Lyumkis - One of the best experts on this subject based on the ideXlab platform.
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model building and refinement of a natively glycosylated hiv 1 env protein by high resolution Cryoelectron Microscopy
Structure, 2015Co-Authors: Dmitry Lyumkis, Andrew B WardAbstract:Summary Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle Cryoelectron Microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 A resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.
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model building and refinement of a natively glycosylated hiv 1 env protein by high resolution Cryoelectron Microscopy
Structure, 2015Co-Authors: Dmitry Lyumkis, Andrew B Ward, Jeong Hyun Lee, Natalia De ValAbstract:Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle Cryoelectron Microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 A resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.