Cryo-electron tomography reveals the binding and release states of the major adhesion complex from Mycoplasma genitalium.

The nap particle is an immunogenic surface adhesion complex from Mycoplasma genitalium. It is essential for motility and responsible for binding sialylated oligosaccharides on the surface of the host cell. The nap particle is composed of two P140-P110 heterodimers, the structure of which was recently solved. However, the interpretation of the mechanism by which the mycoplasma cells orchestrate adhesion remained challenging. Here, we provide cryo-electron tomography structures at ~11 Å resolution, which allow for the distinction between the bound and released state of the nap particle, displaying the in vivo conformational states. Fitting of the atomically resolved structures reveals that bound sialylated oligosaccharides are stabilized by both P110 and P140. Movement of the stalk domains allows for the transfer of conformational changes from the interior of the cell to the binding pocket, thus having the capability of an active release process. It is likely that the same mechanism can be transferred to other Mycoplasma species that belong to the pneumoniae cluster.

Desmosome architecture derived from molecular dynamics simulations and cryo-electron tomography.

Desmosomes are cell-cell junctions that link tissue cells experiencing intense mechanical stress. Although the structure of the desmosomal cadherins is known, the desmosome architecture-which is essential for mediating numerous functions-remains elusive. Here, we recorded cryo-electron tomograms (cryo-ET) in which individual cadherins can be discerned; they appear variable in shape, spacing, and tilt with respect to the membrane. The resulting sub-tomogram average reaches a resolution of ∼26 Å, limited by the inherent flexibility of desmosomes. To address this challenge typical of dynamic biological assemblies, we combine sub-tomogram averaging with atomistic molecular dynamics (MD) simulations. We generate models of possible cadherin arrangements and perform an in silico screening according to biophysical and structural properties extracted from MD simulation trajectories. We find a truss-like arrangement of cadherins that resembles the characteristic footprint seen in the electron micrograph. The resulting model of the desmosomal architecture explains their unique biophysical properties and strength.

Mean curvature motion facilitates the segmentation and surface visualization of electron tomograms.

Three-dimensional visualization of biological samples is essential for understanding their architecture and function. However, it is often challenging due to the macromolecular crowdedness of the samples and low signal-to-noise ratio of the cryo-electron tomograms. Denoising and segmentation techniques address this challenge by increasing the signal-to-noise ratio and by simplifying the data in images. Here, mean curvature motion is presented as a method that can be applied to segmentation results, created either manually or automatically, to significantly improve both the visual quality and downstream computational handling. Mean curvature motion is a process based on nonlinear anisotropic diffusion that smooths along edges and causes high-curvature features, such as noise, to disappear. In combination with level-set methods for image erosion and dilation, the application of mean curvature motion to electron tomograms and segmentations removes sharp edges or spikes in the visualized surfaces, produces an improved surface quality, and improves overall visualization and interpretation of the three-dimensional images.

Structure of RNA polymerase I transcribing ribosomal DNA genes.

RNA polymerase I (Pol I) is a highly processive enzyme that transcribes ribosomal DNA (rDNA) and regulates growth of eukaryotic cells. Crystal structures of free Pol I from the yeast Saccharomyces cerevisiae have revealed dimers of the enzyme stabilized by a 'connector' element and an expanded cleft containing the active centre in an inactive conformation. The central bridge helix was unfolded and a Pol-I-specific 'expander' element occupied the DNA-template-binding site. The structure of Pol I in its active transcribing conformation has yet to be determined, whereas structures of Pol II and Pol III have been solved with bound DNA template and RNA transcript. Here we report structures of active transcribing Pol I from yeast solved by two different cryo-electron microscopy approaches. A single-particle structure at 3.8 Å resolution reveals a contracted active centre cleft with bound DNA and RNA, and a narrowed pore beneath the active site that no longer holds the RNA-cleavage-stimulating domain of subunit A12.2. A structure at 29 Å resolution that was determined from cryo-electron tomograms of Pol I enzymes transcribing cellular rDNA confirms contraction of the cleft and reveals that incoming and exiting rDNA enclose an angle of around 150°. The structures suggest a model for the regulation of transcription elongation in which contracted and expanded polymerase conformations are associated with active and inactive states, respectively.

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.

Cryo-electron tomography analyses of terminal organelle mutants suggest the motility mechanism of Mycoplasma genitalium.

The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron-dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryoelectron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, to systematically analyze the ultrastructural effects. These data were correlated with microcinematographies, from which the motility patterns can be quantitatively assessed. We visualized diverse phenotypes, ranging from mild to severe cell adhesion, motility and segregation defects. Based on our observations, we propose a double-spring ratchet model for the motility mechanism that explains our current and previous observations. Our model, which expands and integrates the previously suggested inchworm model, allocates specific functions to each of the essential components of this unique bacterial motility system.

Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium.

Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo-electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub-tomogram averages of free and cell-attached NAPs respectively, revealing a tetrameric complex with two-fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and ß lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The ß lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative-stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.

Three-dimensional CTF correction improves the resolution of electron tomograms.

Correction of the contrast transfer function (CTF) of the microscope is a necessary step, in order to achieve high resolution from averaged electron microscopic images. Thereby, the CTF is first estimated and subsequently the electron micrograph is corrected, so that the negative oscillations of the CTF are equalized. Typically, the CTF correction is performed in 2D and the tilt-induced focus gradient is taken into account. Most often, the sample-thickness-induced focus gradient is ignored. Theoretical considerations, as well as implementation suggestions, for a 3D CTF correction that considers both gradients have been proposed before, although an implementation achieving a resolution improvement has been lacking, primarily due to computational reasons. Here, we present a comprehensive solution for a 3D CTF correction based on the Jensen-Kornberg scheme, which performs a slice-by-slice correction of the CTF within the tomographic reconstruction. We show that the computational requirements are comparable to those of 2D CTF correction. Using the examples of mitochondrial ribosomes and tobacco mosaic virus we demonstrate the improvement of the reconstruction quality with the 3D CTF correction, and the resolution gain on sub-tomogram averaging. More interestingly, for tomographic applications, the quality of the individual sub-tomograms before averaging increases significantly. We find that 3D CTF correction always produces equal or better results than 2D CTF correction.

Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure.

How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.

M-free: Mask-independent scoring of the reference bias.

The M-free score is a heuristic to measure the reference bias in applications such as template matching and sub-tomogram averaging. In the original formulation the mask typically used in these applications had to be separated into a working and a testing area. Here we present a variant of the calculation of the M-free score, which under certain conditions does not require adapting the mask used during the processing. This is made possible by a modified algorithm that allows for arbitrary variances in the testing and in the working area. Consequently, the reference bias can be estimated with knowledge of only the starting reference, the final average and the mask used for processing. We show that the new formulation of the M-free score gives a reliable measure of the reference bias for any sub-tomogram average that has ancillary data, such as when the averaged structure contains density in the periphery, when a complex is attached to a membrane (membrane-associated complexes) or when one subunit is attached to others (e.g. in viruses). Further, we show that in contrast to correlation-based measurements, the M-free score is sensitive to wrong-alignments and contaminations present in the data set. The scope of this new calculation of the M-free score is to reduce the constraints of the previous approach and in certain cases to avoid an adaptation of the mask. The M-free score gives a separate reliability measure for sub-tomogram averaging and template matching.

Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures.

Yeast prions constitute a "protein-only" mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI(+)], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI(+)] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo-electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced, other cellular complexes, such as ribosomes, were excluded from the fibril arrays. Subtomogram image averaging, made possible by the organized nature of the assemblies, uncovered the presence of an additional array of densities between the fibers. We suggest these structures constitute a self-organizing mechanism that coordinates fiber deposition and the regulation of prion inheritance.

CENP-A arrays are more condensed than canonical arrays at low ionic strength.

The centromeric histone H3 variant centromeric protein A (CENP-A), whose sequence is the least conserved among all histone variants, is responsible for specifying the location of the centromere. Here, we present a comprehensive study of CENP-A nucleosome arrays by cryo-electron tomography. We see that CENP-A arrays have different biophysical properties than canonical ones under low ionic conditions, as they are more condensed with a 20% smaller average nearest-neighbor distance and a 30% higher nucleosome density. We find that CENP-A nucleosomes have a predominantly crossed DNA entry/exit site that is narrowed on average by 8°, and they have a propensity to stack face to face. We therefore propose that CENP-A induces geometric constraints at the nucleosome DNA entry/exit site to bring neighboring nucleosomes into close proximity. This specific property of CENP-A may be responsible for generating a fundamental process that contributes to increased chromatin fiber compaction that is propagated under physiological conditions to form centromeric chromatin.

Super-sampling SART with ordered subsets.

In tomography, the quality of the reconstruction is essential because the complete cascade of the subsequent analysis is based on it. To date, weighted back-projection (WBP) has been the most commonly used technique due to its versatility and performance in sub-tomogram averaging. Here we present super-sampling SART that is based on the simultaneous algebraic reconstruction technique. While algebraic reconstruction techniques typically produce better contrast and lately showed a significant improvement in terms of processing speed, sub-tomogram averages derived from those reconstructions were inferior in resolution compared to those derived from WBP data. Super-sampling SART, however, outperforms both in term of contrast and the resolution achieved in sub-tomogram averaging several other tested methods and in particular WBP. The main feature of super-sampling SART, as the name implies, is the super-sampling option - by which parameter-based up-sampling and down-sampling are used to reduce artifacts. In particular, the aliasing that is omnipresent in the reconstruction can be practically eliminated without a significant increase in the computational time. Furthermore, super-sampling SART reaches convergence within a single iteration, making the processing time comparable to WBP, and eliminating the ambiguity of parameter-controlled convergence times. We find that grouping of projections increases the contrast, while when projections are used individually the resolution can be maximized. Using sub-tomogram averaging of ribosomes as a test case, we show that super-sampling SART achieves equal or better sub-tomogram averaging results than WBP, which is of particular importance in cryo-electron tomography.

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.

Correlative light- and electron microscopy with chemical tags.

Correlative 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 25 nm 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.

Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing.

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei.

Chromatin folding in eukaryotes fits the genome into the limited volume of the cell nucleus. Formation of higher-order chromatin structures attenuates DNA accessibility, thus contributing to the control of essential genome functions such as transcription, DNA replication, and repair. The 30-nm fiber is thought to be the first hierarchical level of chromatin folding, but the nucleosome arrangement in the compact 30-nm fiber was previously unknown. We used cryoelectron tomography of vitreous sections to determine the structure of the compact, native 30-nm fiber of avian erythrocyte nuclei. The predominant geometry of the 30-nm fiber revealed by subtomogram averaging is a left-handed two-start helix with approximately 6.5 nucleosomes per 11 nm, in which the nucleosomes are juxtaposed face-to-face but are shifted off their superhelical axes with an axial translation of approximately 3.4 nm and an azimuthal rotation of approximately 54°. The nucleosomes produce a checkerboard pattern when observed in the direction perpendicular to the fiber axis but are not interdigitated. The nucleosome packing within the fibers shows larger center-to-center internucleosomal distances than previously anticipated, thus excluding the possibility of core-to-core interactions, explaining how transcription and regulation factors can access nucleosomes.

The three-dimensional molecular structure of the desmosomal plaque.

The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a low-density layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.

Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae.

Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.