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PubMed1991

A molecular switch: subunit rotations involved in the right-handed to left-handed transitions of Salmonella typhimurium flagellar filaments.

Using the combined techniques of cryoelectron microscopy and image analysis, we generated three-dimensional reconstructions of flagellar filaments from straight, right-handed (SJW1655-R) and straight, left-handed (SJW1660-L) Salmonella typhimurium mutants, both of which have the same parental strain (SJW1103). In the filaments from SJW1655, all flagellin subunits have the same conformation (R), while in filaments from SJW1660, the subunits are all in the alternate (L) conformation. The difference between the two three-dimensional density maps reveal the structural changes that accompany switching of the flagellin subunits between the two conformations. In going from the R to L state, the subunit undergoes a rotation 30 degrees clockwise about a radial axis and 38 degrees clockwise about a vertical axis, and suffers a 50 degrees bend of the outer, relative to the inner, subunit domain. The intersubunit spacing, along the 11-start protofilaments, changes from 51.6 A in the right-handed filament to 52.1 A in the left-handed filament. In order to produce the correct corkscrew shape in native filaments, the change in contacts that produces this shortening of 0.5 A must occur among the inner domains at a radius of about 30 A. We suggest that the changes in the middle domains of the subunit are the switch that forces changes in the inner domains.

Cell Movement↗

PubMed1991

Structure and assembly of the Escherichia coli transcription termination factor rho and its interaction with RNA. I. Cryoelectron microscopic studies.

Cryoelectron microscopy has been used to visualize the Escherichia coli transcription termination protein rho in a vitreously frozen state, without the use of strains, fixatives or other chemical perturbants. In the absence of RNA cofactor, a variety of structures are observed, reflecting the heterogeneity of complexes formed by rho at protein concentrations near the physiological range (3 to 10 microM). One of the most common structural motifs we see is a six-membered ring of rho subunits (present as either a closed or "notched" circle), which corresponds to the predominant hexameric association state of the protein. Also visible are smaller oligomeric structures, present as curved lines of rho subunits, which probably represent the lower association states of the protein that coexist with the hexamer at these protein concentrations. Addition of oligomers of ribocytosine (rC) of defined lengths (23-mers and 100-mers) results in the generation of more homogeneous populations of rho oligomers. In the presence of (rC)23, all identifiable particles appear either as closed or as notched hexameric circles. A small fraction of these particles are of visibly higher density, and are identified with the dodecamers expected as a subpopulation of rho under these conditions. Binding of (rC)100, an oligomer of length greater than that needed to span the entire hexamer binding site, results in a uniform population of closed circular hexamers. In some images additional features are visible at either the centers or the peripheries of the particles. These features may correspond to the excess length of the rC strands bound to the hexamers. The distributions of particles observed under the various experimental conditions used correlate well to those deduced from physical biochemical studies Seifried et al., accompanying paper).

Bacterial Proteins↗

PubMed1992

Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex.

A specific complex of proteins involved in bacteriophage T4 replication has been visualized by cryoelectron microscopy as distinctive structures in association with DNA. Formation of these structures, which we term "hash-marks" for their characteristic appearance in association with DNA, requires the presence of the T4 polymerase accessory proteins (the products of T4 genes 44, 45 and 62), ATP and appropriate DNA cofactors. ATP hydrolysis by the DNA-stimulated ATPase activity of the accessory proteins is required for visualization of the hash-mark structures. If ATP hydrolysis is stopped by chelation of Mg2+, by dilution with a non-hydrolyzable ATP analogue, or by exhaustion of the ATP supply, the DNA-associated structures disappear within seconds to minutes, indicating that they have a finite and relatively short lifetime. The labile nature of the structures makes their study by more conventional methods of electron microscopy, as well as by most other structural approaches, difficult if not impossible. Addition of T4 gene 32 protein increases the number of hash-mark structures, as well as increasing the rate of ATP hydrolysis. Using plasmid DNA in either a native (supercoiled) or enzymatically modified state, we have shown that nicked or gapped DNA is required as a cofactor for hash-mark formation. Stimulation of the ATPase activity of the accessory proteins has a similar cofactor requirement. These conditions for the formation and visualization of the structures parallel those required for the action of these complexes in promoting the enzymatic activity of the T4 DNA polymerase, as well as the transcription of late T4 genes. Substructure in the hash-marks has been examined by image analysis, which reveals a variation in the projected density of the subunits comprising the structures. The three-dimensional size of the hash-marks, modeled as a solid ellipsoid, is consistent with that of the gene 44/62 protein subcomplex. Density variations suggest an arrangement of subunits, either tetragonal or trigonal, viewed from a variety of angles about the DNA axis. The hash-mark structures often appear in clusters, even in DNA that has a single nick. We interpret this distribution as the result of one-dimensional translocation of the hash-marks along the DNA after their ATP-dependent initial association with, and injection into, the DNA at nicks or gaps.

Adenosine Triphosphatases↗

PubMed1991

Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus.

The organization of DNA within the HSV-1 capsid has been determined by cryoelectron microscopy and image reconstruction. Purified C-capsids, which are fully packaged, were compared with A-capsids, which are empty. Unlike A-capsids, C-capsids show fine striations and punctate arrays with a spacing of approximately 2.6 nm. The packaged DNA forms a uniformly dense ball, extending radially as far as the inner surface of the icosahedral (T = 16) capsid shell, whose structure is essentially identical in A-capsids and C-capsids. Thus we find no evidence for the inner T = 4 shell previously reported by Schrag et al. to be present in C-capsids. Encapsidated HSV-1 DNA closely resembles that previously visualized in bacteriophages T4 and lambda, thus supporting the idea of a close parallelism between the respective assembly pathways of a major family of animal viruses (the herpesviruses) and a major family of bacterial viruses.

Animals↗

PubMed1995

Nucleocapsid and glycoprotein organization in an enveloped virus.

Alphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bilayer, which surrounds the approximately 400 A diameter nucleocapsid, is penetrated by 80 spikes arranged in a T = 4 lattice. Each spike is a trimer of heterodimers consisting of glycoproteins E1 and E2. Cryoelectron microscopy and image reconstruction of Ross River virus showed that the T = 4 quaternary structure of the nucleocapsid consists of pentamer and hexamer clusters of the capsid protein, but not dimers, as have been observed in several crystallographic studies. The E1-E2 heterodimers form one-to-one associations with the nucleocapsid monomers across the lipid bilayer. Knowledge of the atomic structure of the capsid protein and our reconstruction allows us to identify capsid-protein residues that interact with the RNA, the glycoproteins, and adjacent capsid-proteins.

Freezing↗

PubMed1995

Low pH induces swiveling of the glycoprotein heterodimers in the Semliki Forest virus spike complex.

Time-resolved cryoelectron microscopy reveals the first step in the conformational changes that enable membrane fusion in Semliki Forest virus. The neutral pH structure reveals a central cavity within the spike complex, plate-like extensions forming a layer above the membrane, and the paths of the paired transmembrane domains connecting the trimeric spikes and pentamer-hexamer clustered capsid subunits. Low pH treatment results in centrifugal movement of E2, the receptor-binding subunit, centripetal movement of E1 to narrow the central cavity initiating the formation of an E1 trimer, and the extension of the E1 fusion sequence toward the target membrane.

Cryopreservation↗

PubMed1995

Antibody recognition of picornaviruses and escape from neutralization: a structural view.

Escape of picornaviruses from neutralization by monoclonal antibodies is mediated by substitutions of very few, defined amino acid residues of the capsid, generally located on the tip of some surface-exposed loops. Substitutions at the same positions are possibly of major relevance to antigenic variation of picornaviruses in the field. Such residues tend to cluster in discrete areas, termed antigenic sites. The structure of virus-antibody and peptide-antibody complexes, determined by cryoelectron microscopy and X-ray crystallography, combined with studies using site-directed mutagenesis, are beginning to reveal new features of picornavirus epitopes. This information complements and expands the view on picornavirus antigenicity previously provided by analyses of antibody-escape mutants. In addition to amino acids found replaced in escape mutants, other surface residues which remain invariant in spite of immune pressure also participate in contacts with the antibody molecule. Some invariant residues are even critical for the antigen-antibody interaction. Escape mutations occur at the subset of antigenically critical residues which are tolerant to change because they are not essentially involved in capsid structure or function. Restrictions to variation differ among epitopes; this may contribute to explain the different number of serotypes among picornaviruses, and the frequency at which antigenically highly divergent variants occur in the field.

Animals↗

PubMed1992

Ultrastructure of alpha 2-macroglobulins.

New results concerning the ultrastructure of human alpha 2-macroglobulin (alpha 2M) molecules are presented in connection and comparison with the historical, the current and our own most recent, even unpublished results on the structure and function of alpha 2M and related proteins. The electron microscopic approach uses classical negative staining, combined with the new imaging mode "Electron Energy Loss Spectroscopy", which provides unusual contrast, resolution and readability of the electron micrographs. Immuno- and cryoelectron microscopy, as well as image processing has provided new data necessary to the building of tentative 3D models of the molecule. A model for the native tetrameric alpha 2M is described for the first time, and tries to explain and gather the various observations, sometimes contradictory, taken from different laboratories. A revised version for a model of the methylamine- and proteinase-transformed forms of alpha 2M is also shown. The probable positions of the bait regions and the thiol esters are given on both models. We confirm that alpha 2M is a twin trap capable of inactivating one or two proteinases by partial immobilization. Preliminary results on the production of crystals of alpha 2M-chymotrypsin complexes are also presented. A critical analysis of our models is presented in comparison with others. The technical limitations reached with some techniques and some possible extensions of future research in the field are also presented.

Animals↗

PubMed1993

Dissecting the assembly of orbiviruses.

Virus assembly within infected cells involves a precise sequence of macromolecular interactions. To unravel the individual steps involved in the assembly of a complete virion of bluetongue virus, we have engineered a series of recombinant baculoviruses to make multicomponent structures resembling virus structures. When combined with cryoelectron microscopy and image processing techniques the data reveal the organization and assembly of the various components of this virus.

Baculoviridae↗

PubMed1994

Cutting artefacts on ultrathin cryosections of biological bulk specimens.

Cryoelectron microscopy of ultrathin cyrosections permits study of the ultrastructure of cells in the hydrated state. However, cryosectioning deforms the cut volume as the sections are compressed in the cutting direction while their thickness is increased with respect to the cutting feed. The compression has been measured on the elliptical deformation of lipid droplets in liver cells. It is about 40% at 48 degrees cutting angle while it is reduced to 30% at 38 degrees. The section deformation involves the underlying ultrastructure as far as the specimen is deformable. Three deformation modes are discussed. Crystalline ice is broken into chips which are tilted and pressed out of the section plane to account for the section deformation. Within one chip, the ultrastructure of the ice is preserved. In contrast, hydrated catalase crystals deform plastically. Cutting shortens the crystal distances the same amount as cryosections are compressed. Distances in hexagonal DNA crystals are also influenced by cutting since the spots of diffraction patterns are blurred in the cutting direction. However, the crystal spacing is not shortened in the cutting direction. The crystal apparently reorganizes during cutting deformation.

Animals↗

PubMedDate not supplied

Three-dimensional reconstruction of a complex of human alpha 2-macroglobulin with monomaleimido Nanogold (Au1.4nm) embedded in ice.

Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the alpha 2M-Nanogold conjugates (alpha 2M-Au1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed alpha 2M molecules. From this study we propose three possible locations for the cysteine 949.

Cysteine↗

PubMed2004

Testing the geometric clutch hypothesis.

The Geometric Clutch hypothesis is based on the premise that transverse forces (t-forces) acting on the outer doublets of the eukaryotic axoneme coordinate the action of the dynein motors to produce flagellar and ciliary beating. T-forces result from tension and compression on the outer doublets when a bend is present on the flagellum or cilium. The t-force acts to pry the doublets apart in an active bend, and push the doublets together when the flagellum is passively bent and thus could engage and disengage the dynein motors. Computed simulations of this working mechanism have reproduced the beating pattern of simple cilia and flagella, and of mammalian sperm. Cilia-like beating, with a clearly defined effective and recovery stroke, can be generated using one uniformly applied switching algorithm. When the mechanical properties and dimensions appropriate to a specific flagellum are incorporated into the model the same algorithm can simulate a sea urchin or bull sperm-like beat. The computed model reproduces many of the observed behaviors of real flagella and cilia. The model can duplicate the results of outer arm extraction experiments in cilia and predicted two types of arrest behavior that were verified experimentally in bull sperm. It also successfully predicted the experimentally determined nexin elasticity. Calculations based on live and reactivated sea urchin and bull sperm yielded a value of 0.5 nN/microm for the t-force at the switch-point. This is a force sufficient to overcome the shearing force generated by all the dyneins on one micron of outer doublet. A t-force of this magnitude should produce substantial distortion of the axoneme at the switch-point, especially in spoke or spoke-head deficient motile flagella. This concrete and verifiable prediction is within the grasp of recent advances in imaging technology, specifically cryoelectron microscopy and atomic force microscopy.

Animals↗

PubMed2005

Spectroscopic studies on native and protofibrillar insulin.

The structure of insulin in amyloid fibrillar form has been recently shown as a well folded conformation using cryoelectron microscopy [Jimenez, J.L., Nettleton, E.J., Bouchard, M., Robinson, C.V., Dobson, C.M., Saibil H.R., 2002. The protofilament structure of insulin amyloid fibrils. Proc. Natl. Acad. Sci. USA. 99 9196-9201.]. Most of the amyloid aggregates elicit maximum toxicity in the protofibrillar (PF) intermediate state. Here, we describe PF intermediates of insulin are made-up monomers with flexible conformers. We also show protofibrils have three-dimensionally extended hydrophobic cavity to bind with 1-anilinonaphthalene-8-sulphonate (ANS) molecules. Energy transfer measurement revealed that ANS dye binding site of PF is within the range of FRET distance of insulin tyrosine residues. Significant proportion of beta-sheet, helical, and turn structures in the PF form indicate conformational dynamics in the folded chain of insulin in the PF assembled form. Though the conformational flexibility is noticeably present in the assembly, addition of GdnHCl could completely unfold PF into disordered structure suggesting structural "zipping" in the PF form. We have also shown that helical conformer inducing solvent 2,2,2-trifluoroethanol (TFE) could dissociate the PF aggregate indicating possible involvement of beta-sheets in contributing to PF stability.

Amyloid↗

PubMed2006

Proteolytic mapping of the adeno-associated virus capsid.

The three-dimensional structures of the viral capsid of three AAV serotypes have previously been determined by X-ray crystallography or cryoelectron microscopy. These studies of AAV and similar studies of autonomous parvoviruses have yielded important structural information about the virions in a low-energy conformation. However, there is little information on the structural properties of AAV virions in solution under physiological conditions. We demonstrate that proteolytic digestion of AAV2 virions with trypsin results in cleavage at a specific site on the capsid surface while the capsid remains intact. The products of digestion were mapped using unique antibodies, protein sequencing, mass spectroscopy, and 3D structure modeling to a region on a surface loop that is common to all three AAV2 structural proteins. Empty AAV2 capsids could be distinguished from full (DNA-containing) capsids, having an increased susceptibility of VP2 to trypsin and being digested more rapidly by chymotrypsin. Proteolytic analysis utilizing trypsin or chymotrypsin was also capable of distinguishing AAV2 from AAV1 and AAV5, as seen by differential susceptibility and unique fragment patterns. These data demonstrate a novel approach for studying the structure of AAV capsids in solution and should be valuable in the testing and engineering of AAV vectors for gene transfer.

Amino Acid Sequence↗

PubMed2001

Na(+)/H(+) antiporters.

Na(+)/H(+) antiporters are membrane proteins that play a major role in pH and Na(+) homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants. The emerging genomic sequence projects already have started to reveal that the Na(+)/H(+) antiporters cluster in several families. Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na(+)/H(+) antiporter of Escherichia coli. This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes. It has recently been crystallized in both 3D as well as 2D crystals. The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 A resolution was obtained and a 3D map was reconstructed. NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing. This is the first insight into the structure of a polytopic membrane protein. Many Na(+)/H(+) antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis. The molecular mechanism underlying this pH sensitivity has been studied in NhaA. Amino acid residues involved in the pH response have been identified. Conformational changes transducing the pH change into a change in activity were found in loop VIII-IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively. Regulation by pH of the eukaryotic Na(+)/H(+) antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am. J. Physiol. 269 (1995) G1-G11). The transcription of NhaA has been shown to be regulated by a novel Na(+)-specific regulatory network. It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters.

Amino Acid Sequence↗

PubMed1986

Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I.

Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.

Crystallization↗

PubMed1990

Visualization of transport-related configurations of the nuclear pore transporter.

The transport of macromolecules between the cytoplasm and nucleus of the cell is mediated by the nuclear pore complex (NPC). In this study, details of the central transporter assembly within NPCs have been examined by cryoelectron microscopy, image processing, and classification analysis. The NPC transporter in isolated amphibian nuclei appears to adopt a minimum of four transport-related configurations including: (a) a putative closed form with a 90-100 A diameter central pore, (b) a docked form with material aligned over the pore, (c) an open form with substrates apparently caught "in transit," and (d) an open form with an enlarged pore. This data confirms previous observations on NPC transporters labeled with nucleoplasmin-gold (Akey, C.W., and D.S. Goldfarb. 1989. J. Cell Biol. 109:971-982) and allows a working model of the central NPC transporter to be proposed. The model is comprised of two supramolecular irislike assemblies which open asynchronously to provide an expanded pore for translocation while maintaining transport fidelity.

Animals↗

PubMed1995

Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles.

The morphological consequences of differences in the monolayer surface areas of large unilamellar vesicles (LUVs) have been examined employing cryoelectron microscopy techniques. Surface area was varied by inducing net transbilayer transport of dioleoylphosphatidylglycerol (DOPG) in dioleoylphosphatidylcholine (DOPC):DOPG (9:1, mol:mol) LUVs in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner to the outer monolayer, initially invaginated LUVs are transformed to long narrow tubular structures, or spherical structures with one or more protrusions. Tubular structures are also seen in response to outward DOPG transport in DOPC:DOPG:Chol (6:1:3, mol:mol:mol) LUV systems, and when lyso-PC is allowed to partition into the exterior monolayer of DOPC:DOPG (9:1, mol:mol) LUVs in the absence of DOPG transport. Conversely, when the inner monolayer area is expanded by the transport of DOPG from the outer monolayer to the inner monolayer of non-invaginated LUVs, a reversion to invaginated structures is observed. The morphological changes are well described by an elastic bending theory of the bilayer. Identification of the difference in relaxed monolayer areas and of the volume-to-area ratio of the LUVs as the shape-determining factors allows a quantitative classification of the observed morphologies. The morphology seen in LUVs supports the possibility that factors leading to differences in monolayer surface areas could play important roles in intracellular membrane transport processes.

Cholesterol↗