A semisynthetic peptide-metalloporphyrin responsive matrix for artificial photosynthesis

Solid state NMR/Biophysical Organic Chemistr

Formation and structure of a NAIP5-NLRC4 inflammasome induced by direct interactions with conserved N- and C-terminal regions of flagellin

The NOD-like receptors NAIP5 and NLRC4 play an essential role in the innate immune response to the bacterial tail protein flagellin. Upon flagellin detection, NAIP5 and NLRC4 form a hetero-oligomeric inflammasome that induces caspase-1-dependent cell death. So far, both the mechanism of formation of the NAIP5-NLRC4 inflammasome and its structure are poorly understood. In this study we combine inflammasome reconstitution in HEK293 cells, purification of inflammasome components, and negative stain electron microscopy to address these issues. We find that a Salmonella typhimurium flagellin fragment comprising the D0 domain and the neighboring spoke region is able to co-precipitate NAIP5 and induce formation of the NAIP5-NLRC4 inflammasome. Comparison with smaller fragments indicates that flagellin recognition is mediated by its C-terminal residues as well as the spoke region. We reconstitute the inflammasome from purified flagellin, NAIP5, and NLRC4, thus proving that no other cellular components are required for its formation. Electron micrographs of the purified inflammasome provide unprecedented insight into its architecture, revealing disk-like complexes consisting of 11 or 12 protomers in which NAIP5 and NLRC4 appear to occupy equivalent positions. On the basis of our data, we propose a model for inflammasome formation wherein direct interaction of flagellin with a single NAIP5 induces the recruitment and progressive incorporation of NLRC4, resulting in the formation of a hetero-oligomeric inflammasome

Феномен самодостатності містико-естетичного досвіду: місце в розумінні подібності християнства, даосизму, релігії давніх українців і сучасного містицизму

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells

Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium

Microbial Biotechnolog

Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium

The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall ofStreptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down oftagOexpression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that theStreptomycescell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.Eveline Ultee et al. reveal the architecture of the actinobacterial cell wall of a polar growing bacteriumStreptomyces coelicolor, using cryo-electron tomography. This study suggests that theStreptomycescell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.Microbial Biotechnolog

Structural studies by cryo-electron tomography of two initiation complexes of the immune system: C1-IgG6 and NAIP5-NLRC4

Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and the killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy. Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from invading microorganisms activate NLRs and induce NLR multimerization. Little is known about the conformational changes involved in NLR activation and the structural organization of NLR multimers. Here we show by cryo-electron tomography that flagellin-induced NAIP5/NLRC4 multimers form right- and left-handed helical polymers with a diameter of 28 nm and a rise of 6.5 nm. Sub tomogram averaging produced an electron density map at 4 nm resolution, which was used for rigid body fitting of NLR subdomains derived from the crystal structure of dormant NLRC4. The resulting structural model of inflammasome-incorporated NLRC4 indicates that a prominent rotation of the LRR domain of NLRC4 is necessary for multimer formation, providing unprecedented insight in the conformational changes that accompany NLR activation. The resolution of electron tomograms is anisotropic due to geometrical constraints during data collection, such as the limited tilt range and single-axis tilt series acquisition. Acquisition of dual axis-tilt series can decrease these effects. However, in cryo-electron tomography, to limit the electron radiation damage that occurs during imaging, the total dose should not increase and must be fractionated over the two tilt series. Here, we set out to determine whether it is beneficial to fractionate electron dose for recording dual-axis cryo-electron tilt series or whether it is better to perform single-axis acquisition. To assess the quality of tomographic reconstructions in different directions, we introduce conical Fourier shell correlation (cFSCe/o). Employing cFSCe/o, we compared the resolution isotropy of single-axis and dual-axis (cryo-)electron tomograms using even/odd split data sets. We show that the resolution of dual-axis simulated and cryo-electron tomograms in the plane orthogonal to the electron beam becomes more isotropic compared to single-axis tomograms and high-resolution peaks along the tilt axis disappear. cFSCe/o also allowed us to compare different methods for the alignment of dual-axis tomograms. We show that different tomographic reconstruction programs produce varying anisotropic resolution patterns in dual- axis tomograms. We anticipate that cFSCe/o can also be useful for comparisons of acquisition and reconstruction parameters, and different hardware implementations

From tube to structure: SPA Cryo-EM workflow using apoferritin as an example

In this chapter, we present an overview of a standard protocol to achieve structure determination at high resolution by Single Particle Analysis cryogenic Electron Microscopy using apoferritin as a standard sample. The purified apoferritin is applied to a glow-discharged support and then flash frozen in liquid ethane. The prepared grids are loaded into the electron microscope and checked for particle spreading and ice thickness. The microscope alignments are performed and the data collection session is setup for an overnight data collection. The collected movies containing two-dimensional images of the apoferritin sample are then processed to obtain a high-resolution three-dimensional reconstruction

Pushing the resolution limits in cryo electron tomography of biological structures

Microscopic imaging and technolog

Conical Fourier shell correlation applied to electron tomograms

The resolution of electron tomograms is anisotropic due to geometrical constraints during data collection, such as the limited tilt range and single axis tilt series acquisition. Acquisition of dual axis tilt series can decrease these effects. However, in cryo-electron tomography, to limit the electron radiation damage that occurs during imaging, the total dose should not increase and must be fractionated over the two tilt series. Here we set out to determine whether it is beneficial fractionate electron dose for recording dual axis cryo electron tilt series or whether it is better to perform single axis acquisition. To assess the quality of tomographic reconstructions in different directions here we introduce conical Fourier shell correlation (cFSCe/o). Employing cFSCe/o, we compared the resolution isotropy of single-axis and dual-axis (cryo-)electron tomograms using even/odd split data sets. We show that the resolution of dual-axis simulated and cryo-electron tomograms in the plane orthogonal to the electron beam becomes more isotropic compared to single-axis tomograms and high resolution peaks along the tilt axis disappear. cFSCe/o also allowed us to compare different methods for the alignment of dual-axis tomograms. We show that different tomographic reconstruction programs produce different anisotropic resolution in dual axis tomograms. We anticipate that cFSCe/o can also be useful for comparisons of acquisition and reconstruction parameters, and different hardware implementations

Structure of C1-antibody complexes determined by cryo-electron tomography

Microscopic imaging and technolog