Single Particle Image Analysis Research Articles

Cryo-Electron Microscopy Structure of Human Peroxiredoxin-3 Filament Reveals the Assembly of a Putative Chaperone

Peroxiredoxins (Prxs) are a ubiquitous class of thiol-dependent peroxidases that play an important role in the protection and response of cells to oxidative stress. The catalytic unit of typical 2-Cys Prxs are homodimers, which can self-associate to form complex assemblies that are hypothesized to have signaling and chaperone activity. Mitochondrial Prx3 forms dodecameric toroids, which can further stack to form filaments, the so-called high-molecular-weight (HMW) form that has putative holdase activity. We used single-particle analysis and helical processing of electron cryomicroscopy images of human Prx3 filaments induced by low pH to generate a ∼7-Å resolution 3D structure of the HMW form, the first such structure for a 2-Cys Prx. The pseudo-atomic model reveals interactions that promote the stacking of the toroids and shows that unlike previously reported data, the structure can accommodate a partially folded C terminus. The HMW filament lumen displays hydrophobic patches, which we hypothesize bestow holdase activity.

Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

Dye lipophilicity and retention in lipid membranes: implications for single-molecule spectroscopy.

Fluorescence studies of individual lipid vesicles rely on the proper positioning of probes in the lipid milieu. This is true for both positional tags and chemoselective fluorogenic probes that undergo chemical modification following reaction with an analyte of interest within the lipid environment. The present report describes lipophilicity and localization estimations for a series of BODIPY dyes bearing substituents of varying hydrophobicity. We also studied fluorogenic trap-reporter probes that undergo fluorescence emission enhancement upon trapping of reactive oxygen species (ROS), including lipid peroxyl radicals. We show that caution has to be taken to extrapolate ensemble partition measurements of dyes to the single-molecule regime as a result of the dramatically different lipid concentration prevailing in ensemble versus single-molecule experiments. We show that the mole fraction of dyes that remains embedded in liposomes during a typical single-molecule experiment may be accurately determined from a ratiometric single-particle imaging analysis. We further demonstrate that fluorescence correlation spectroscopy (FCS) provides a very rapid and reliable estimate of the lipophilic nature of a given dye under highly dilute single-molecule-like conditions. Our combined single-particle spectroscopy and FCS experiments suggest that the minimal mole fraction of membrane-associated dyes (x(m)) as determined from FCS experiments is about 0.5 for adequate dye retention during single-molecule imaging in lipid membranes. Our work further highlights the dramatic effect that chemical modifications can have on chemoselective fluorogenic probe localization.

Peroxiredoxin is a versatile self-assembling tecton for protein nanotechnology.

The potential for protein tectons to be used in nanotechnology is increasingly recognized, but the repertoire of stable proteins that assemble into defined shapes in response to an environmental trigger is limited. Peroxiredoxins (Prxs) are a protein family that shows an amazing array of supramolecular assemblies, making them attractive tectons. Human Prx3 (hPrx3) forms toroidal oligomers characteristic of the Prx family, but no structure has been solved to date. Here we report the first 3-D structure of this protein, derived from single-particle analysis of TEM images, establishing a dodecameric structure. This result was supported by SAXS measurements. We also present the first detailed structure of a double toroidal Prx from a higher organism determined by SPA. Guided by these structures, variants of the protein were designed to facilitate controlled assembly of protein nanostructures through the association of the toroids. We observed an enhanced population of stacked toroids, as seen by TEM; nanocages and interlocked toroids were also visible. Low pH was successfully predicted to generate long ordered nanotubes. Control over the length of the tubes was gained by adding ammonium sulfate to the assembly buffer. These versatile assembly properties demonstrate the considerable potential of hPrx3 as a tecton for protein nanotechnology.

Zebrafish Cardiac Muscle Thick Filaments: Isolation Technique and Three-Dimensional Structure

To understand how mutations in thick filament proteins such as cardiac myosin binding protein-C or titin, cause familial hypertrophic cardiomyopathies, it is important to determine the structure of the cardiac thick filament. Techniques for the genetic manipulation of the zebrafish are well established and it has become a major model for the study of the cardiovascular system. Our goal is to develop zebrafish as an alternative system to the mammalian heart model for the study of the structure of the cardiac thick filaments and the proteins that form it. We have successfully isolated thick filaments from zebrafish cardiac muscle, using a procedure similar to those for mammalian heart, and analyzed their structure by negative-staining and electron microscopy. The isolated filaments appear well ordered with the characteristic 42.9 nm quasi-helical repeat of the myosin heads expected from x-ray diffraction. We have performed single particle image analysis on the collected electron microscopy images for the C-zone region of these filaments and obtained a three-dimensional reconstruction at 3.5 nm resolution. This reconstruction reveals structure similar to the mammalian thick filament, and demonstrates that zebrafish may provide a useful model for the study of the changes in the cardiac thick filament associated with disease processes.

Structure of the Kidney Slit Diaphragm Adapter Protein CD2-Associated Protein as Determined with Electron Microscopy

CD2-associated protein (CD2AP) is a multidomain scaffolding protein that has a critical role in renal function. CD2AP is expressed in glomerular podocytes at the slit diaphragm, a modified adherens junction that comprises the protein filtration barrier of the kidney, and interacts with a number of protein ligands involved in cytoskeletal remodeling, membrane trafficking, cell motility, and cell survival. The structure of CD2AP is unknown. We used electron microscopy and single particle image analysis to determine the three-dimensional structure of recombinant full-length CD2AP and found that the protein is a tetramer in solution. Image reconstruction of negatively stained protein particles generated a structure at 21 Å resolution. The protein assumed a roughly spherical, very loosely packed structure. Analysis of the electron density map revealed that CD2AP consists of a central coiled-coil domain, which forms the tetramer interface, surrounded by four symmetry-related motifs, each containing three globular domains corresponding to the three SH3 domains. The spatial organization exposes the binding sites of all 12 SH3 domains in the tetramer, allowing simultaneous binding to multiple targets. Determination of the structure of CD2AP provides novel insights into the biology of this slit diaphragm protein and lays the groundwork for characterizing the interactions between key molecules of the slit diaphragm that control glomerular filtration.

Iterative Elastic 3D-to-2D Alignment Method Using Normal Modes for Studying Structural Dynamics of Large Macromolecular Complexes

This article presents a method to study large-scale conformational changes by combining electron microscopy (EM) single-particle image analysis and normal mode analysis (NMA). It is referred to as HEMNMA, which stands for hybrid electron microscopy normal mode analysis. NMA of a reference structure (atomic-resolution structure or EM volume) is used to predict possible motions that are then confronted with EM images within an automatic iterative elastic 3D-to-2D alignment procedure to identify actual motions in the imaged samples. HEMNMA can be used to extensively analyze the conformational changes and may be used in combination with classic discrete procedures. The identified conformations allow modeling of deformation pathways compatible with the experimental data. HEMNMA was tested with synthetic and experimental data sets of E.coli 70S ribosome, DNA polymerase Pol α and B subunit complex of the eukaryotic primosome, and tomato bushy stunt virus.

Abstract P3-05-16: Quantitative diagnostic technology for HER2 positive breast cancer by molecular imaging: The potential predictive factor of anti-HER2 therapy

Abstract Background: Anti-HER2 therapy contributes to improvement of the clinical outcome in HER2-positive patients. However, current studies have reported the resistance to trastuzumab. To diagnose HER2 expression level, immunohistochemical (IHC) method with 3,3’-diaminobenzidine (IHC-DAB) staining is widely used, but is not quantitative and classifies tumors into only 4 categories. Additionally, the epitopes of trastuzumab and most antibodies used for IHC-DAB are different. These problems might induce a gap between diagnostics and therapeutic efficacy. To improve the method for cancer patients’ HER2 status, we developed a novel IHC method with trastuzumab conjugated with fluorescent nano-particles quantum dots (QDs) by single-particle imaging using tissue samples from patients with clinical information. Methods: We prepared two kinds of QD-conjugated antibody complexes for IHC with QDs (IHC-QD). One was a QD-conjugated trastuzumab, the other was a QD-conjugated human IgG for control. We performed the several steps of antigen-antibody reaction by two kinds of QD-conjugated antibody probes for 37 human breast cancer specimens. The QD signal reflected HER2 expression level was observed by the optical system that consisted of an epi-fluorescent microscope with EM-CCD camera. Results: We successfully obtained the images of only QDs signal by the image analyses, and calculated the number of QD particles in a cell and defined it as IHC-QD score that reflects the level of HER2 protein expression in cancer cells. In comparing IHC-QD scores with IHC-DAB scores, cases with a score of 0 and 1 by IHC-DAB had low scores of IHC-QD (0.01-0.67), being in narrow range. On the other hand, those with a score of 2 varied widely, with IHC-QD scores of 0.06 to 16.98, and those with a score of 3 also varied, from 0.18 to 18.81. In some of the sample with IHC-DAB score of 2 and 3, the binding of trastuzumab to these cancer cells is very weak although they are candidate for trastuzumab therapy in present HER2 diagnostic criteria. In comparison between FISH and IHC-QD scores, the straight-line approximation curve shows that both quantitative scores, FISH (0.96-8.76) and IHC-QD (0.01-18.81) are well correlated (R = 0.83, p<0.001). In analyzing the therapeutic efficacy in 14 metastatic breast cancers treated with trastuzumab according to IHC-QD or FISH or IHC-DAB scores, IHC-QD score was correlated with time to progression (TTP) more than FISH and IHC-DAB scores. Table 1Diagnostic methodsPatient numberTime to progression, median(range)P valueIHC-DAB 0.207Score 236 (3-9) Score 31112 (3-60) FISH 0.577Low score712 (5-60) High score79 (3-52) IHC-QD 0.094Low score76 (3-60) High score724 (3-52) Comparison between IHC-QD or FISH or IHC-DAB and the therapeutic efficacy with trastuzumab Althogh the sample size was small, this result suggests that the IHC-QD score has a potential role as predictive factor for trastuzumab therapy. Conclusion: We performed the precisely quantitative IHC using trastuzumab-conjugated QDs and single-particle imaging analysis, and propose using IHC-QD as a predictive factor for trastuzumab therapy. This new diagnostic method would be expected to contribute to the development of a therapeutic strategy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-16.

Structural Insight into Torque Generation Mechanism of the Bacterial Flagellar Motor

The bacterial flagellum is made of a rotary motor and a long helical filament by means of which bacteria swim. The flagellar motors of Salmonella and E. coli rotate at around 300 Hz and drive rapid rotation of each flagellum to propel cell movements. The mechanism of torque generation by the motor has been intensively studied by many groups based on the torque-speed relationships and the structure, but it has been elusive due to the lack of high-resolution structural information of the rotor and stator and their interactions. X-ray crystal structures are available for some part of the rotor and switch complex component proteins but no detailed information is yet available for their arrangement in the motor assembly and also for the transmembrane and cytoplasmic domains of the stator. A currently proposed 3D model of the rotor for torque generation does not satisfy the stepping behavior of the motor observed by single motor nanophotometry. We have been trying to solve the structure of the flagellar hook-basal body by electron cryomicroscopy and single particle image analysis in its isolated form from the membrane as well as by electron cryotomography of the cell to visualize the basal body in situ. I will report our current progress and discuss some structural insights into the torque generation mechanism.

3D-Reconstruction of the Isolated Zebrafish Cardiac Muscle Thick Filament

The zebrafish is an important model for studying the development of the cardiovascular system and the effect of mutations in various cardiovascular genes. Because of the relative ease of genetic screening, the zebrafish provides a unique model system for the study of the location and functional role of the proteins in adult macromolecular structures such as the cardiac thick filament. As a step towards this goal, we successfully developed a technique for the isolation of zebrafish cardiac thick filaments and examined them by negative staining with uranyl acetate and electron microscopy (EM). The isolated filaments appear well ordered with the characteristic 43 nm quasi-helical repeat of the myosin heads. We have also performed single particle image analysis on the collected EM images of the C-zone region of these filaments and obtained a 3D-reconstruction at 35A resolution. This 3D-reconstruction shows many similarities, but also some differences from previous reconstructions of mammalian cardiac thick filaments. Our goal is to further refine the current image map of the zebrafish cardiac thick filament and use it as a model for comparison with the structure of isolated thick filaments with mutations or modifications in the thick filament associated proteins. Supported by a NIH grant SC1HL096017 to RWK and a British Heart Foundation Fellowship FS/07/017/22951 to HA.

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

The L-type Ca(2+) channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α(1s) or Ca(V)1.1, α(2), β(1a), δ1, and γ), we created transgenic mice expressing a recombinant β(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α(2)-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca(V)1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α(1s) subunit to the membrane and its suggested role in excitation-contraction coupling.

Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-D-maltoside (β-DM) or n-dodecyl-α-D-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.

Interaction of HLA‐DR and CD74 at the cell surface of antigen‐presenting cells by single particle image analysis

Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.

3-Dimensional Structure of Human Cardiac Muscle Myosin Filaments by Electron Microscopy and Single Particle Analysis

In all muscles the interaction between actin and myosin filaments leads to muscle contraction and force production mediated by the hydrolysis of ATP. Actin filament structure is well understood to high resolution, but myosin filament structure is much less well defined. Myosin filaments are formed from complicated arrangements of myosin molecules and accessory proteins (e.g. C-protein [MyBP-C] and titin). We compare the arrangement of the myosin heads in the different species and how these arrangements change in diseased muscle. We have developed a method to solve the 3-dimensional (3D) structure of myosin filaments by single particle analysis of electron microscope (EM) data. We already defined the 3D structure of myosin filaments of various muscles from different species by both X-ray diffraction modelling and EM and single particle analysis including insect flight muscle, scallop striated muscle, fish skeletal muscle and rabbit cardiac muscle. We are now studying the 3D structure of myosin filaments isolated from human heart muscles.Mutations in cardiac myosin, C-protein and titin are known to be associated with cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand myosin-associated heart disease, it is important to understand the 3D structure of myosin filaments in normal heart muscle.Recently we have developed procedures to isolate human cardiac muscle myosin filaments preserving their highly ordered pseudo-helical structure thus making them amenable, for the first time, to EM and single particle image analysis. We have collected EM data from myosin filaments isolated from both normal and failing hearts, and have so far processed the data from normal heart muscle. Analysis of the 3D structure of myosin filaments in normal heart muscle will permit the structural effects of known myosin filaments-associated mutations to be investigated in detail.

Collagen VI, Conformation of A-domain Arrays and Microfibril Architecture

Collagen VI is a ubiquitous extracellular matrix protein that assembles into beaded microfibrils that form networks linking cells to the matrix. Collagen VI microfibrils are typically formed from a heterotrimer of the α1, α2, and α3 chains. The α3 chain is distinct as it contains an extended N terminus with up to 10 consecutive von Willebrand factor type A-domains (VWA). Here, we use solution small angle x-ray scattering (SAXS) and single particle analysis EM to determine the nanostructure of nine of these contiguous A-domains. Both techniques reveal a tight C-shape conformation for the A-domains. Furthermore, using biophysical approaches, we demonstrate that the N-terminal region undergoes a conformational change and a proportion forms dimers in the presence of Zn2+. This is the first indication that divalent cations interact with collagen VI A-domains. A three-dimensional reconstruction of tissue-purified collagen VI microfibrils was generated using EM and single particle image analysis. The reconstruction showed the intricate architecture of the collagen VI globular regions, in particular the highly structurally conserved C-terminal region and variations in the appearance of the N-terminal region. The N-terminal domains project out from the globular beaded region like angled radial spokes. These could potentially provide interactive surfaces for other cell matrix molecules.

Optical analysis of particle size and chromite liberation from pulp samples of a UG2 ore regrinding circuit

Since the early seventies high chromite and low base metal sulphide (BMS) contents of the UG2 reef imposes technological challenges to mineral processors and extractive metallurgists. Forty years later, particle size distribution and size by size mineralogy are considered as key factors to the continuous improvement of the UG-2 ore metallurgy. With the successful development of ultra fine grinding technologies, a compromise has to be found between fine grinding to achieve platinum group minerals liberation and to avoid the overgrinding of gangue minerals, especially chromite gangue which is detrimental in smelting process. Indeed, fine chromite grains, despite of being naturally hydrophobic mineral, can be recovered in flotation concentrate by entrainment. In addition, overgrinding also increase liberation of naturally floatable gangue (talc) which contaminate the concentrate and need to be controlled during flotation. In this paper, innovative techniques of single particle image analysis from both dry and wet samples have been tentatively used to assess the chromite particle size distribution and the chromite grade of samples taken from a secondary milling circuit. Preliminary results demonstrate excellent potential for online particle imaging making use of both the particle geometry (size, shape) and the optical properties (translucency).

Structural Analysis of Cytoplasmic Dynein Tail Domain

Cytoplasmic dynein is microtubule-based motor protein utilizing the energy from ATP hydrolysis and plays fundamental roles in various cellular activities including mitosis, vesicle transport and cell migration. Cytoplasmic dynein is a huge protein complex consisting of the head domain and the tail domain. The head domain consists of six AAA modules and a stalk. So far, many studies have investigated about the head domain, because it contains ATP hydrolysis and motor activities. On the other hand, the tail domain consists of N-terminal third of heavy chains (HC), intermediate chains (IC), light intermediate chains (LIC), and light chains (LC). Although the tail domain is implicated in binding of cargos and some of dynein binding proteins which control motor activities of dynein, for example dynactin, the detailed molecular architecture of the tail domain remains to be unveiled. In this study, we observed cytoplasmic dynein molecules with or without polyhistidine tag (His-tag) by transmission electron microscopy (TEM), performed single particle image analysis and obtained the averaged image of the tail domain. Using nickel nitrilotriacetic acid (Ni-NTA) conjugated gold nanoparticles to label His-tag of recombinant proteins, we identified the positions of N-terminus and C-terminus of IC and determined its orientation in the tail domain. Furthermore, we identified the position of C-terminus of LIC relative to IC. Based on our observations, we propose a new model of the architecture of the dynein tail domain which differs from currently advocating model.

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c(10) ring and the other stator subunits at the F(0)-F(0) dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356-12358). In the ATP synthase dimer, the two peripheral stalks are located near the F(1)-F(1) interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.

Dual-axis cryo-electron tomography on intact mitochondria reveals the association of ATP synthase into oligomeric chains

Cryo-electron tomography (CET) is an emerging electron microscopic technique for reconstructing a 3D volume representing large non-periodic objects, such as organelles, under cryogenic “life-like” conditions by incrementally tilting the specimen. Until recently only single-axis CET was possible due to technical limitations, but recently a rotation stage was developed allowing to collect two orthogonal tilt series in a cryo-protected way. Dual-axis tilting reduces the missing information of sampling originating from single-axis tilting over restricted tilt angles. In spite of that up to now most of the work on whole mitochondria is done by chemical fixation and plastic embedding. We implemented dual-axis cryo-electron tomography on an FEI Polara microscope and use it to generate tomograms from the frozen-hydrated mitochondria at about 6 nm resolution. The overall organisation of mitochondrial proteins remains a puzzle, therefore we initiated our work on intact mitochondria from Bos taurus and Polytomella, a green alga related to Chlamydomonas. We have obtained 3D reconstructions of intact mitochondria by cryo-ET and averaged subvolumes of oligomeric ATP synthase at 5.7 nm resolution [1]. This revealed the 3D arrangement of rows of ATP synthase dimers within the cristae membranes. Specific known details of monomeric ATP synthase, such as the connection of the stator to the OSCP subunit, were revealed for the first time in intact organelles. Previously performed single particle image analysis on isolated dimeric ATP synthases revealed a 70° angle between monomers, which strongly pointed out their functional role in the curvature of the inner mitochondrial membrane. The measured angle of 70° between the two F1FO ATP synthases in intact mitochondria supports this assumption. In fragmented bovine mitochondrial membranes dimers are flexible and the angle ranged from 55° to 95° [1]. Rendering of the membrane surfaces clearly demonstrated rows of ATP synthases located mostly in the curved region of the lamella cristae in bovine mitochondria and along the whole surface of tubular cristae in Polytomella. Likely other OXPHOS complexes are located between the rows of ATP synthases in Polytomella whereas the less highly curved regions are rather empty, as it was proposed for mammalian mitochondria [2].

Structure of the 100S Ribosome in the Hibernation Stage Revealed by Electron Cryomicroscopy

In the stationary growth phase of bacteria, protein biosynthesis on ribosomes is suppressed, and the ribosomes are preserved in the cell by the formation of the 100S ribosome. The 100S ribosome is a dimer of the 70S ribosome and is formed by the binding of the ribosome modulation factor and the hibernation promoting factor. However, the binding mode between the two 70S ribosomes and the mechanism of complex formation are still poorly understood. Here, we report the structure of the 100S ribosome by electron cryomicroscopy and single-particle image analysis. The 100S ribosome purified from the cell in the stationary growth phase is composed of two transfer RNA-free 70S ribosomes, has two-fold symmetry, and is formed through interactions between their 30S subunits, where interactions between small subunit proteins, S2, S3 and S5, appear to be critical for the dimerization.