Lactose binding to heat-labile enterotoxin revealed by X-ray crystallography.

Recognition of the oligosaccharide portion of ganglioside GM1 in membranes of target cells by the heat-labile enterotoxin from Escherichia coli is the crucial first step in its pathogenesis, as it is for the closely related cholera toxin. These toxins have five B subunits, which are essential for GM1 binding, and a single A subunit, which needs to be nicked by proteolysis and reduced, yielding an A1-'enzyme' and an A2-'linker' peptide. A1 is translocated across the membrane of intestinal epithelial cells, possibly after endocytosis, upon which it ADP-ribosylates the G protein Gs alpha. The mechanism of binding and translocation of these toxins has been extensively investigated, but how the protein is orientated on binding is still not clear. Knowing the precise arrangement of the ganglioside binding sites of the toxins will be useful for designing drugs against the diarrhoeal diseases caused by organisms secreting these toxins and in the development of oral vaccines against them. We present here the three-dimensional structure of the E. coli heat-labile enterotoxin complexed with lactose. This reveals the location of the binding site of the terminal galactose of GM1, which is consistent with toxin binding to the target cell with its A1 fragment pointing away from the membrane. A small helix is identified at the carboxy terminus of A2 which emerges through the central pore of the B subunits and probably comes into contact with the membrane upon binding, whereas the A1 subunit is flexible with respect to the B pentamer.     

Pheromone binding to two rodent urinary proteins revealed by X-ray crystallography.

The principal protein excreted in male rat urine, urinary alpha 2-globulin and the homologous mouse protein, major urinary protein, have been well characterized, although their functions remain unclear. Male rat urine affects the behaviour and sexual response of female rats, leading to the proposal that rodent urinary proteins are responsible for binding pheromones and their subsequent release from drying urine. Urinary alpha 2-globulin is also involved in hyaline droplet nephropathy, an important toxicological syndrome in male rats resulting from exposure to a number of industrial chemicals and characterized by the accumulation of liganded urinary alpha 2-globulin in lysosomes in the kidney, followed by the induction of renal cancer. We now report the three-dimensional structures of mouse major urinary protein (at 2.4 A resolution) and rat urinary alpha 2-globulin (at 2.8 A resolution). The results corroborate the role of these proteins in pheromone transport and elaborate the structural basis of ligand binding.     

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography.

Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 A resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few ?ngstroms by fitting atomic models of CPMV4 and Fab (Kol)5 to reconstructed density maps.     

Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions

The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.     

Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy

The protein cyclophilin is the major intracellular receptor for the immunosuppressive drug cyclosporin A. Cyclosporin A acts as an inhibitor of T-cell activation and can prevent graft rejection in organ and bone marrow transplantation. Cyclophilin may be responsible for mediating this immunosuppressive response. Cyclophilin also catalyses the interconversion of the cis and trans isomers of the peptidyl-prolyl amide bonds of peptide and protein substrates. Here we report the X-ray crystal structure of human recombinant cyclophilin complexed with a tetrapeptide and the identification, by nuclear magnetic resonance spectroscopy, of the specific binding site for cyclosporin A. Cyclophilin has an eight-stranded antiparallel beta-barrel structure. The prolyl isomerase substrate-binding site is coincident with the cyclosporine-binding site. These results may help to provide a structural basis for rationalizing the immunosuppressive function of the cyclosporin-cyclophilin system and will also be important in the design of improved immunosuppressant drugs.     

X-ray crystallography of the binding of the bacterial cell wall trisaccharide NAM-NAG-NAM to lysozyme

Hen egg white lysozyme was the first enzyme whose structure was determined by X-ray crystallography. The proposed mechanism based on this structure involves the distortion of the saccharide residue (2-acetamido-2-deoxy-D-muramic acid, NAM) in the natural substrate (an alternating beta (1 leads to 4) linked oligomer of 2-acetamido-2-deoxy-D-glucose (NAG) and NAM residues) bound to site D in the binding cleft. The importance of substrate distortion has prompted numerous enzymatic, chemical, theoretical, and physical studies, but there is little direct crystallographic evidence on the conformation of a NAM residue bound at site D. We now present the X-ray structure of the non-hydrolysed trisaccharide NAM-NAG-NAM bound in subsites B, C, D. Our interpretation of the 2.5-A resolution difference map does not involve distortion of this residue in site D. Comparison with the structure of the delta-lactone derived from tetra N-acetylchitotetraose (NAG)3NAL) bound to lysozyme suggests we may be looking at a Michaelis complex.     

Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.     

A common mammalian plan of accessory optic system organization revealed in all primates

The accessory optic system (AOS), which was described as early as 1870 by Gudden, constitutes a distinct midbrain visual pathway in all classes of vertebrates. In non-primate mammals, retinal fibres of this system project to a set of three nuclei: the dorsal (DTN), the lateral (LTN) and the medial (MTN) terminal nuclei. Whereas all AOS cells respond to the slow motion of large visual stimuli, the neurons are tuned to complementary directions of movement: horizontal temporo-nasal direction for the DTN, vertical up and down for the LTN and vertical down for the MTN. It has thus been suggested that these nuclei establish a system of retinal coordinates for the detection of whole field motion. As the AOS provides direct and indirect pathways to both oculomotor and vestibular structures, each of these nuclei is thought to be an essential link in the co-ordination of eye and head movements in relation to movement within the visual-field. One problem for the generalization of this theory is that the medial terminal nucleus has never been found in primates. In this report we establish both the existence of this nucleus and its afferent input from the retina in all major groups of primates (prosimians, New and Old World monkeys and apes), indicating a common anatomical plan of organization of the AOS in mammals.     

Three-dimensional structure of plant light-harvesting complex determined by electron crystallography

The structure of the light-harvesting chlorophyll a/b-protein complex, a membrane protein serving as the major antenna of solar energy in plant photosynthesis, has been determined at 6 A resolution by electron crystallography. Within the complex, three membrane-spanning alpha helices and 15 chlorophyll molecules are resolved. There is an intramolecular diad relating two of the alpha helices and some of the chlorophylls. The spacing of the chlorophylls suggests energy transfer by delocalized exciton coupling and F?rster mechanisms.     

Architecture of ribonucleoprotein complexes in influenza A virus particles

In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10-15 nm in width and 30-120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.     

Genome organization and transactivation of the human immunodeficiency virus type 2

Analysis of the nucleotide sequence of the human retrovirus associated with AIDS in West Africa, HIV-2, shows that it is evolutionarily distant from the previously characterized HIV-1. We suggest that these viruses existed long before the current AIDS epidemics. Their biological properties are conserved in spite of limited sequence homology; this may help the determination of the structure-function relationships of the different viral elements.     

Structural architecture of an outer membrane channel as determined by electron crystallography.

Porins are a family of membrane channels commonly found in the outer membranes of Gram-negative bacteria where they serve as diffusional pathways for waste products, nutrients and antibiotics, and can also be receptors for bacteriophages. Porin channels have been shown in vitro to be voltage-gated. They can exhibit slight selectivities for certain solutes; for example PhoE porin has some selectivity for anionic and phosphate-containing compounds. Unlike many known membrane proteins which often contain long stretches of hydrophobic segments that are believed to traverse the membrane in a helical conformation, porins are found to have charged residues distributed almost uniformly along their primary sequences and have most of their secondary structure in a beta-sheet conformation. We have made crystalline patches of PhoE porin embedded in a lipid bilayer and have used these to determine the structure of PhoE porin by electron crystallography to a resolution of 6A. The basic structure consists of a trimer of elliptically shaped, cylindrical walls of beta sheet. Each cylinder has an inner lining, formed by parts of the polypeptide, that defines the channel size. The structure provides a clue as to how deletions of segments of polypeptide, which are found in certain mutants, can result in an actual increase in the channel size.     

Biosignatures as revealed by spectropolarimetry of Earthshine

Low-resolution intensity spectra of Earth's atmosphere obtained from space reveal strong signatures of life ('biosignatures'), such as molecular oxygen and methane with abundances far from chemical equilibrium, as well as the presence of a 'red edge' (a sharp increase of albedo for wavelengths longer than 700?nm) caused by surface vegetation. Light passing through the atmosphere is strongly linearly polarized by scattering (from air molecules, aerosols and cloud particles) and by reflection (from oceans and land). Spectropolarimetric observations of local patches of Earth's sky light from the ground contain signatures of oxygen, ozone and water, and are used to characterize the properties of clouds and aerosols. When applied to exoplanets, ground-based spectropolarimetry can better constrain properties of atmospheres and surfaces than can standard intensity spectroscopy. Here we report disk-integrated linear polarization spectra of Earthshine, which is sunlight that has been first reflected by Earth and then reflected back to Earth by the Moon. The observations allow us to determine the fractional contribution of clouds and ocean surface, and are sensitive to visible areas of vegetation as small as 10 per cent. They represent a benchmark for the diagnostics of the atmospheric composition, mean cloud height and surfaces of exoplanets.     

Gate-tuning of graphene plasmons revealed by infrared nano-imaging

Surface plasmons are collective oscillations of electrons in metals or semiconductors that enable confinement and control of electromagnetic energy at subwavelength scales. Rapid progress in plasmonics has largely relied on advances in device nano-fabrication, whereas less attention has been paid to the tunable properties of plasmonic media. One such medium--graphene--is amenable to convenient tuning of its electronic and optical properties by varying the applied voltage. Here, using infrared nano-imaging, we show that common graphene/SiO(2)/Si back-gated structures support propagating surface plasmons. The wavelength of graphene plasmons is of the order of 200 nanometres at technologically relevant infrared frequencies, and they can propagate several times this distance. We have succeeded in altering both the amplitude and the wavelength of these plasmons by varying the gate voltage. Using plasmon interferometry, we investigated losses in graphene by exploring real-space profiles of plasmon standing waves formed between the tip of our nano-probe and the edges of the samples. Plasmon dissipation quantified through this analysis is linked to the exotic electrodynamics of graphene. Standard plasmonic figures of merit of our tunable graphene devices surpass those of common metal-based structures.     

The internal structure of Early Cambrian fossil embryo Olivooides revealed in the light of Synchrotron X-ray Tomographic Microscopy

Countless fossil embryo Olivooides and the hatched larvae,juveniles and adults(the latter two kinds are Punctatus) are recovered by means of acid maceration from the fine-crystalline to medium-crystalline phosphatic limestone and phosphatic micrite of Early Cambrian Kuanchuanpu Formation at the Shizhonggou section,near Kuanchuanpu Village,Ningqiang County,Shaanxi Province,China.Using the technique of Synchrotron X-ray Tomographic Microscopy,the 3D internal structure of Olivooides and Punctatus is reconstructed.The morphological and statistic analyses are also given to the stellae structure of Olivooides and Punctatus,which indicates that this structure is a result of adaptive evolution to a lifestyle of fast-attaching after hatching,probably with the function of mucilage secretion.The internal structure of Punctatus is described and discussed.The ovum-like structure,a common internal feature of Punctatus,is considered as the taphonomic structure,rather than eggs or other biological structure.This structure is thought to be formed after the burial of the animal and before or during the mineralization.The original internal structure of Punctatus is assumed to be tabulae-filled,with soft body grown on them.