The reaction centre of the photounit of Rhodospirillum rubrum is anchored to the light-harvesting complex with four-fold rotational disorder

The minimal photounit of the photosynthetic membranes of the purple non-sulphur bacterium Rhodospirillum rubrum, comprising the reaction centre and the light-harvesting complex has been purified and crystallised in two dimensions in the presence of added phospholipids, and subsequently visualised by electron microscopy after negatively-staining. The position of the reaction centres within the light-harvesting ring has been determined at low resolution by the application of a new analysis for rotationally disordered identical units (here the reaction centres) within a two-dimensional crystalline lattice comprised of perfectly aligned unit cells (here the light-harvesting complexes). The reaction centre was found to preferentially occupy one of four orientations within the light-harvesting complex. The light-harvesting complex appears to be distorted to C4 symmetry, thus assuming a squarish shape when visualised by negative staining. A tentative structural model of the reaction centre-light-harvesting complex photounit which fits the experimental data is propose

Protocols for Subtomogram Averaging of Membrane Proteins in the Dynamo Software Package

Cryo-electron tomography allows low-resolution three-dimensional (3D) viewing of cellular organelles and macromolecular complexes present as multiple copies within a tomogram. These structures are computationally extracted and averaged in order to obtain high-resolution 3D structures, and provide a map of their spatial distribution and interaction with their biological microenvironment. To do so, we apply the user-friendly Dynamo software package on a tomographic data set. Dynamo acts as a modular toolbox adaptable to different biological scenarios, allowing a custom designed pipeline for subtomogram averaging. Here, we use as a textbook example the mitochondrial docking site of the positive-strand RNA Flock house nodavirus (FHV) to describe how Dynamo coordinates several basic steps in the subtomogram averaging workflow. Our framework covers specific strategies to deal with additional issues in subtomogram averaging as tomographic data management, 3D surface visualization, automatic assignment of asymmetry and inherent loss of Fourier information in presence of preferential views

Cryo-EM structure of native human thyroglobulin

The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T; 3; ) and thyroxine (T; 4; ) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations

Coordination-Driven Monolayer-to-Bilayer Transition in 2D Metal-Organic Networks

We report on monolayer-to-bilayer transitions in 2D metal−organic networks (MONs) from amphiphiles supported at the water−air interface. Functionalized calix[4]arenes are assembled through the coordination of selected transition metal ions to yield monomolecular 2D crystalline layers. In the presence of Ni(II) ions, interfacial self-assembly and coordination yields stable monolayers. Cu(II) promotes 2D coordination of a monolayer which is then diffusively reorganizing, nucleates, and grows a progressive amount of second layer islands. Atomic force microscopic data of these layers after transfer onto solid substrates reveal crystalline packing geometries with submolecular resolution as they are varying in function of the building blocks and the kinetics of the assembly. We assign this monolayer-to- bilayer transition to a diffusive reorganization of the initial monolayers owing to chemical vacancies of the predominant coordination motif formed by Cu2+ ions. Our results introduce a new dimension into the controlled monolayer-to-multilayer architecturing of 2D metal− organic networks

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure–frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a “canonical” IFT motor, whereas OSM-3 is an “accessory” IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death