Structure of the type VI secretion system contractile sheath.

Bacteria use rapid contraction of a long sheath of the type VI secretion system (T6SS) to deliver effectors into a target cell. Here, we present an atomic-resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four ß strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.

KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in Plasmodium falciparum-infected erythrocytes.

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

Structure of RyR1 in native membranes.

Ryanodine receptor 1 (RyR1) mediates excitation-contraction coupling by releasing Ca2+ from sarcoplasmic reticulum (SR) to the cytoplasm of skeletal muscle cells. RyR1 activation is regulated by several proteins from both the cytoplasm and lumen of the SR. Here, we report the structure of RyR1 from native SR membranes in closed and open states. Compared to the previously reported structures of purified RyR1, our structure reveals helix-like densities traversing the bilayer approximately 5 nm from the RyR1 transmembrane domain and sarcoplasmic extensions linking RyR1 to a putative calsequestrin network. We document the primary conformation of RyR1 in situ and its structural variations. The activation of RyR1 is associated with changes in membrane curvature and movement in the sarcoplasmic extensions. Our results provide structural insight into the mechanism of RyR1 in its native environment.

Structure of the Yersinia injectisome in intracellular host cell phagosomes revealed by cryo FIB electron tomography.

Many pathogenic bacteria use the type III secretion system (T3SS), or injectisome, to secrete toxins into host cells. These protruding systems are primary targets for drug and vaccine development. Upon contact between injectisomes and host membranes, toxin secretion is triggered. How this works structurally and functionally is yet unknown. Using cryo-focused ion beam milling and cryo-electron tomography, we visualized injectisomes of Yersinia enterocolitica inside the phagosomes of infected human myeloid cells in a close-to-native state. We observed that a minimum needle length is required for injectisomes to contact the host membrane and bending of host membranes by some injectisomes that contact the host. Through subtomogram averaging, the structure of the entire injectisome was determined, which revealed structural differences in the cytosolic sorting platform compared to other bacteria. These findings contribute to understanding how injectisomes secrete toxins into host cells and provides the indispensable native context. The application of these cryo-electron microscopy techniques paves the way for the study of the 3D structure of infection-relevant protein complexes in host-pathogen interactions.

De novo protein structure determination from near-atomic-resolution cryo-EM maps.

We present a de novo model-building approach that combines predicted backbone conformations with side-chain fit to density to accurately assign sequence into density maps. This method yielded accurate models for six of nine experimental maps at 3.3- to 4.8-Å resolution and produced a nearly complete model for an unsolved map containing a 660-residue heterodimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic-resolution cryo-electron microscopy (cryo-EM) maps.

Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.

Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal "needle complex" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.

Dynamo Catalogue: Geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms.

Cryo electron tomography allows macromolecular complexes within vitrified, intact, thin cells or sections thereof to be visualized, and structural analysis to be performed in situ by averaging over multiple copies of the same molecules. Image processing for subtomogram averaging is specific and cumbersome, due to the large amount of data and its three dimensional nature and anisotropic resolution. Here, we streamline data processing for subtomogram averaging by introducing an archiving system, Dynamo Catalogue. This system manages tomographic data from multiple tomograms and allows visual feedback during all processing steps, including particle picking, extraction, alignment and classification. The file structure of a processing project file structure includes logfiles of performed operations, and can be backed up and shared between users. Command line commands, database queries and a set of GUIs give the user versatile control over the process. Here, we introduce a set of geometric tools that streamline particle picking from simple (filaments, spheres, tubes, vesicles) and complex geometries (arbitrary 2D surfaces, rare instances on proteins with geometric restrictions, and 2D and 3D crystals). Advanced functionality, such as manual alignment and subboxing, is useful when initial templates are generated for alignment and for project customization. Dynamo Catalogue is part of the open source package Dynamo and includes tools to ensure format compatibility with the subtomogram averaging functionalities of other packages, such as Jsubtomo, PyTom, PEET, EMAN2, XMIPP and Relion.

Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence.

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by the actions of Rho-glucosylating toxins A and B. Recently identified hypervirulent strains, which are associated with increased morbidity and mortality, additionally produce the actin-ADP-ribosylating toxin C. difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here we show that CDT-induced protrusions allow vesicle traffic and contain endoplasmic reticulum tubules, connected to microtubules via the calcium sensor Stim1. The toxin reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, from basolateral to the apical membrane sides in a microtubule- and Stim1-dependent manner. The data yield a model of C. difficile adherence regulated by actin depolymerization, microtubule restructuring, subsequent Stim1-dependent Ca(2+) signaling, vesicle rerouting, and secretion of ECM proteins to increase bacterial adherence.

Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation.

Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high-resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo-electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca(2+) ), the intensity of single spots significantly increased compared with non-secreting conditions (presence of Ca(2+) ), in line with an overall up-regulation of expression levels of all components. Single injectisomes observed by cryo-electron tomography tended to cluster at distances less than 100 nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up-regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells.

Electron tomography of Plasmodium falciparum merozoites reveals core cellular events that underpin erythrocyte invasion.

Erythrocyte invasion by merozoites forms of the malaria parasite is a key step in the establishment of human malaria disease. To date, efforts to understand cellular events underpinning entry have been limited to insights from non-human parasites, with no studies at sub-micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub-cellular compartments during infectionincomplete, in particular that of the secretory organelles. Using advances in P. falciparum merozoite isolation and new imaging techniques we present a three-dimensional study of invasion using electron microscopy, cryo-electron tomography and cryo-X-ray tomography. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion.

Interaction of complexes I, III, and IV within the bovine respirasome by single particle cryoelectron tomography.

The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III(2), and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III(2) and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.

Structural characterization of Thogoto Virus nucleoprotein provides insights into viral RNA encapsidation and RNP assembly.

Orthomyxoviruses, such as influenza and thogotoviruses, are important human and animal pathogens. Their segmented viral RNA genomes are wrapped by viral nucleoproteins (NPs) into helical ribonucleoprotein complexes (RNPs). NP structures of several influenza viruses have been reported. However, there are still contradictory models of how orthomyxovirus RNPs are assembled. Here, we characterize the crystal structure of Thogoto virus (THOV) NP and found striking similarities to structures of influenza viral NPs, including a two-lobed domain architecture, a positively charged RNA-binding cleft, and a tail loop important for trimerization and viral transcription. A low-resolution cryo-electron tomography reconstruction of THOV RNPs elucidates a left-handed double helical assembly. By providing a model for RNP assembly of THOV, our study suggests conserved NP assembly and RNA encapsidation modes for thogoto- and influenza viruses.

Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites.

Microtubules are dynamic cytoskeletal structures important for cell division, polarity, and motility and are therefore major targets for anticancer and antiparasite drugs. In the invasive forms of apicomplexan parasites, which are highly polarized and often motile cells, exceptionally stable subpellicular microtubules determine the shape of the parasite, and serve as tracks for vesicle transport. We used cryoelectron tomography to image cytoplasmic structures in three dimensions within intact, rapidly frozen Plasmodium sporozoites. This approach revealed microtubule walls that are extended at the luminal side by an additional 3 nm compared to microtubules of mammalian cells. Fourier analysis revealed an 8-nm longitudinal periodicity of the luminal constituent, suggesting the presence of a molecule interacting with tubulin dimers. In silico generation and analysis of microtubule models confirmed this unexpected topology. Microtubules from extracted sporozoites and Toxoplasma gondii tachyzoites showed a similar density distribution, suggesting that the putative protein is conserved among Apicomplexa and serves to stabilize microtubules.

Environmental constraints guide migration of malaria parasites during transmission.

Migrating cells are guided in complex environments mainly by chemotaxis or structural cues presented by the surrounding tissue. During transmission of malaria, parasite motility in the skin is important for Plasmodium sporozoites to reach the blood circulation. Here we show that sporozoite migration varies in different skin environments the parasite encounters at the arbitrary sites of the mosquito bite. In order to systematically examine how sporozoite migration depends on the structure of the environment, we studied it in micro-fabricated obstacle arrays. The trajectories observed in vivo and in vitro closely resemble each other suggesting that structural constraints can be sufficient to guide Plasmodium sporozoites in complex environments. Sporozoite speed in different environments is optimized for migration and correlates with persistence length and dispersal. However, this correlation breaks down in mutant sporozoites that show adhesion impairment due to the lack of TRAP-like protein (TLP) on their surfaces. This may explain their delay in infecting the host. The flexibility of sporozoite adaption to different environments and a favorable speed for optimal dispersal ensures efficient host switching during malaria transmission.

Structural basis for chirality and directional motility of Plasmodium sporozoites.

Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent-shaped sporozoites move on 2D substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non-motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo-electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As a consequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli.

Cryo-electron tomography reveals four-membrane architecture of the Plasmodium apicoplast.

BACKGROUND: The apicoplast is a plastid organelle derived from a secondary endosymbiosis, containing biosynthetic pathways essential for the survival of apicomplexan parasites. The Toxoplasma apicoplast clearly possesses four membranes but in related Plasmodium spp. the apicoplast has variably been reported to have either three or four membranes. METHODS: Cryo-electron tomography was employed to image merozoites of Plasmodium falciparum and Plasmodium berghei frozen in their near-native state. Three-dimensional reconstructions revealed the number of apicoplast membranes and the association of the apicoplast with other organelles. Routine transmission electron microscopy of parasites preserved by high-pressure freezing followed by freeze substitution techniques was also used to analyse apicoplast morphology. RESULTS: Cryo-preserved parasites showed clearly four membranes surrounding the apicoplast. A wider gap between the second and third apicoplast membranes was frequently observed. The apicoplast was found in close proximity to the nucleus and to the rhoptries. The apicoplast matrix showed ribosome-sized particles and membranous whorls. CONCLUSIONS: The Plasmodium apicoplast possesses four membranes, as do the apicoplasts of other apicomplexan parasites. This is consistent with a four-membraned secondary endosymbiotic plastid ancestor.

A weak-labelling and deep learning approach for in-focus object segmentation in 3D widefield microscopy.

Three-dimensional information is crucial to our understanding of biological phenomena. The vast majority of biological microscopy specimens are inherently three-dimensional. However, conventional light microscopy is largely geared towards 2D images, while 3D microscopy and image reconstruction remain feasible only with specialised equipment and techniques. Inspired by the working principles of one such technique-confocal microscopy, we propose a novel approach to 3D widefield microscopy reconstruction through semantic segmentation of in-focus and out-of-focus pixels. For this, we explore a number of rule-based algorithms commonly used for software-based autofocusing and apply them to a dataset of widefield focal stacks. We propose a computation scheme allowing the calculation of lateral focus score maps of the slices of each stack using these algorithms. Furthermore, we identify algorithms preferable for obtaining such maps. Finally, to ensure the practicality of our approach, we propose a surrogate model based on a deep neural network, capable of segmenting in-focus pixels from the out-of-focus background in a fast and reliable fashion. The deep-neural-network-based approach allows a major speedup for data processing making it usable for online data processing.

Streamlined structure determination by cryo-electron tomography and subtomogram averaging using TomoBEAR.

Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR-a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent ("white box") approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available.

Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments.

Dynamo is a new software package for subtomogram averaging of cryo Electron Tomography (cryo-ET) data with three main goals: first, Dynamo allows user-transparent adaptation to a variety of high-performance computing platforms such as GPUs or CPU clusters. Second, Dynamo implements user-friendliness through GUI interfaces and scripting resources. Third, Dynamo offers user-flexibility through a plugin API. Besides the alignment and averaging procedures, Dynamo includes native tools for visualization and analysis of results and data, as well as support for third party visualization software, such as Chimera UCSF or EMAN2. As a demonstration of these functionalities, we studied bacterial flagellar motors and showed automatically detected classes with absent and present C-rings. Subtomogram averaging is a common task in current cryo-ET pipelines, which requires extensive computational resources and follows a well-established workflow. However, due to the data diversity, many existing packages offer slight variations of the same algorithm to improve results. One of the main purposes behind Dynamo is to provide explicit tools to allow the user the insertion of custom designed procedures - or plugins - to replace or complement the native algorithms in the different steps of the processing pipeline for subtomogram averaging without the burden of handling parallelization. Custom scripts that implement new approaches devised by the user are integrated into the Dynamo data management system, so that they can be controlled by the GUI or the scripting capacities. Dynamo executables do not require licenses for third party commercial software. Sources, executables and documentation are freely distributed on http://www.dynamo-em.org.

Assessing the benefits of focal pair cryo-electron tomography.

Cryo electron tomography provides nanometer-scale information on biological matter preserved in a close-to native state. The resolution of tomograms and structures resolved by sub-tomogram averaging is typically limited by the contrast transfer function of the electron microscope, which is especially critical for thick samples. Here, we report a method to increase the attainable resolution by recording tomographic 'focal pairs', which are pairs of tilt series of the same object acquired in complementary defocus conditions. Low defocus imaging provides high resolution at low contrast, while high defocus imaging yields high contrast at the price of limited resolution. Quantitative assessment of the quality of lipid bilayer reconstructions in the resulting tomograms demonstrates stable resolution preservation beyond 3 nm for cells thicker than 500 nm. Further, in computational simulations on synthetic datasets we show the applicability of the method to sub-tomogram averaging, demonstrating its potential for achieving higher resolution.