3D ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography

The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology

Correlative Light- and Electron Microscopy with chemical tags

AbstractCorrelative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells

Cysteine oxidation and disulfide formation in the ribosomal exit tunnel.

Funder: DFG graduate college: CLiC State of Hesse HMWK: BMRZUnderstanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding

Essential protein P116 extracts cholesterol and other indispensable lipids for Mycoplasmas

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically extracts lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are extracted. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake.We thank L. Company and I. Fernández-Vidal for their support during MALS and mass spectroscopy measurements, A. Iborra (Servei de Cultius Cellulars, Anticossos Citometria, UAB) for his assistance with immunizing mice, D. Santos for his assistance in the radioactivity experiment and R. Pérez-Luque and D. Aparicio for their constant support and discussions. J. P. was funded by grants BIO2017-84166-R and PID2021-125632OB-C22 from the ministerio de Ciencia, Innovación y Universidades (MICINN, Spain). I. F. was funded by MICINN-Spain grant PID2021-125632OB-C21. A. S. F. was supported by the Deutsche Forschungsgemeinschaft (FR 1653/14-1 for MS and, FR 1653/6-3 for LS) and the Research Training Group iMOL (GRK 2566/1 for SM)

It’s noisy out there! A review of denoising techniques in cryo-electron tomography

Cryo-electron tomography is the only technique that can provide sub-nanometer resolved images of cell regions or even whole cells, without the need of labeling or staining methods. Technological advances over the past decade in electron microscope stability, cameras, stage precision and software have resulted in faster acquisition speeds and considerably improved resolution. In pursuit of even better image resolution, researchers seek to reduce noise – a crucial factor affecting the reliability of the tomogram interpretation and ultimately limiting the achieved resolution. Sub-tomogram averaging is the method of choice for reducing noise in repetitive objects. However, when averaging is not applicable, a trade-off between reducing noise and conserving genuine image details must be achieved. Thus, denoising is an important process that improves the interpretability of the tomogram not only directly but also by facilitating other downstream tasks, such as segmentation and 3D visualization. Here, I review contemporary denoising techniques for cryo-electron tomography by taking into account noise-specific properties of both reconstruction and detector noise. The outcomes of different techniques are compared, in order to help researchers select the most appropriate for each dataset and to achieve better and more reliable interpretation of the tomograms

M-free : scoring the reference bias in sub-tomogram averaging and template matching

Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells

Ex vivo visualization of RNA polymerase III-specific gene activity with electron microscopy

The direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors

Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro.

International audienceIn this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo

Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

International audienceAlthough the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt

Toward detecting and identifying macromolecules in a cellular context: Template matching applied to electron tomograms

Electron tomography is the only technique available that allows us to visualize the three-dimensional structure of unfixed and unstained cells currently with a resolution of 6–8 nm, but with the prospect to reach 2–4 nm. This raises the possibility of detecting and identifying specific macromolecular complexes within their cellular context by virtue of their structural signature. Templates derived from the high-resolution structure of the molecule under scrutiny are used to search the reconstructed volume. Here we outline and test a computationally feasible two-step procedure: In a first step, mean-curvature motion is used for segmentation, yielding subvolumes that contain with a high probability macromolecules in the expected size range. Subsequently, the particles contained in the subvolumes are identified by cross-correlation, using a set of three-dimensional templates. With simulated and real tomographic data we demonstrate that such an approach is feasible and we explore the detection limits. Even structurally similar particles, such as the thermosome, GroEL, and the 20S proteasome can be identified with high fidelity. This opens up exciting prospects for mapping the territorial distribution of macromolecules and for analyzing molecular interactions in situ