Understanding the apparent stator-rotor connections in the rotary ATPase family using coarse-grained computer modeling

Advances in structural biology, such as cryo-electron microscopy (cryo-EM) have allowed for a number of sophisticated protein complexes to be characterized. However, often only a static snapshot of a protein complex is visualized despite the fact that conformational change is frequently inherent to biological function, as is the case for molecular motors. Computer simulations provide valuable insights into the different conformations available to a particular system that are not accessible using conventional structural techniques. For larger proteins and protein complexes, where a fully atomistic description would be computationally prohibitive, coarse-grained simulation techniques such as Elastic Network Modeling (ENM) are often employed, whereby each atom or group of atoms is linked by a set of springs whose properties can be customized according to the system of interest. Here we compare ENM with a recently proposed continuum model known as Fluctuating Finite Element Analysis (FFEA), which represents the biomolecule as a viscoelastic solid subject to thermal fluctuations. These two complementary computational techniques are used to answer a critical question in the rotary ATPase family; implicit within these motors is the need for a rotor axle and proton pump to rotate freely of the motor domain and stator structures. However, current single particle cryo-EM reconstructions have shown an apparent connection between the stators and rotor axle or pump region, hindering rotation. Both modeling approaches show a possible role for this connection and how it would significantly constrain the mobility of the rotary ATPase family

Rotating with the brakes on and other unresolved features of the vacuolar ATPase

The rotary ATPase family is comprised of the ATP synthase (F-ATPase), vacuolar ATPase (V-ATPase) and acrahael ATPase (A-ATPase). These either predominantly utilise a proton gradient for ATP synthesis or use ATP to produce a proton gradient, driving secondary transport and acidifying organelles. With advances in electron microscopy (EM) has come a significant increase in our understanding of the rotary ATPase family. Following the sub nm resolution reconstructions of both the F and V-ATPase the secondary structure organisation of the elusive subunit a has now been resolved, revealing a novel helical arrangement. Despite these significant developments in our understanding of the rotary ATPases there are still a number of unresolved questions about the mechanism, regulation, and overall architecture, which this mini-review aims to highlight and discuss

Nanotomographie en mode STEM-HAADF (application aux nanomatériaux)

La tomographie électronique est une technique utilisée pour caractériser en 3D la structure et la chimie des matériaux, avec une résolution nanométrique dans le cas d'un microscope électronique par transmission. Le mode d'imagerie choisi est le champ sombre annulaire à grand angle, car il est adapté à la tomographie quantitative à la fois pour les échantillons cristallins et non-cristallins. De plus, le contraste en champ sombre annulaire dépend de la nature chimique des éléments observés, et la simulation des images permet d'extraire des informations chimiques, comme la densité volumique ou le numéro atomique des espèces chimiques présentes. L'objectif de cette thèse est triple: (i) dans un premier temps, adapter le microscope électronique par transmission (MET) à émission de champ du CLYM (Centre Lyonnais de Microscopie) à la tomographie par rotation, (ii) ensuite, appliquer cette approche à l'étude de nanostructures hétérogènes ainsi que de nanomatériaux, (iii) finalement, explorer des méthodes 3D alternatives, comme la stéréoscopie, qui nécessite l'acquisition d'un nombre plus faible d'images qu'en tomographie électronique. Le travail expérimental a consisté à adapter l'embout du porte objet du MET, afin d'atteindre une plage de tilt au delà de 160 : une expérience de tomographie nécessite l'acquisition d'une centaine d'images sur différentes inclinaisons. Un logiciel a été développé pour contrôler semi-automatiquement le microscope et les conditions d'utilisation du détecteur, notamment la correction du focus dans les images durant la phase d'acquisition. Différents matériaux ont été étudiés: des nanoprécipités de carbure de vanadium (VC), des nanoparticules de catalyseurs (Pd), des nanocomposites de type "Au@SiOx" et un alliage présentant une nano-précipitation (AlZnMg).Electron tomography is a technique used to characterise 3D structure and chemistry of the observed samples, with a nanometer resolution when applied in a Transmission Electron Microscope. The chosen imaging mode is STEM-HAADF (Scanning Transmission Electron Microscopy in the High Angle Annular Dark Field imaging mode) because it is well-adapted to a quantitative tomography, for both crystalline and amorphous materials. Moreover the STEM HAADF contrast is related to the chemical nature of elements, and simulation of images can be undertaken to extract chemical information, such as volume density or atomic number of particles. The aim of this thesis is threefold: (i) firstly to adapt the transmission electron microscope of the laboratory to tilting tomography, (ii) secondly to apply this approach to the study of heterogeneous nanostructures and nanomaterials, (iii) endly to explore alternative 3D methods, such as extended stereoscopy, which requires the acquisition of fewer images as compared to complete titlitng tomography . The experimental work has consisting in adaptating the tip of the TEM specimen holder in order to reach a tilt range up of 160, as a tomography experience requires acquisition of hundreds of images at different tilt. A software has been written to control semi-automatically the microscope and the detector, and especially to correct the focus in images during the phase of acquisition. The materials which have been studied are: nanoprecipitates of VC, Pd catalysts, Au@SiOx nanocomposites, and an AlZnMg alloy.VILLEURBANNE-DOC'INSA LYON (692662301) / SudocSudocFranceF

STEM-HAADF nanotomography: application to nanomaterials

La tomographie électronique est une technique utilisée pour caractériser en 3D la structure et la chimie des matériaux, avec une résolution nanométrique dans le cas d\u27un microscope électronique par transmission. Le mode d\u27imagerie choisi est le champ sombre annulaire à grand angle, car il est adapté à la tomographie quantitative à la fois pour les échantillons cristallins et non-cristallins. De plus, le contraste en champ sombre annulaire dépend de la nature chimique des éléments observés, et la simulation des images permet d\u27extraire des informations chimiques, comme la densité volumique ou le numéro atomique des espèces chimiques présentes. L\u27objectif de cette thèse est triple: (i) dans un premier temps, adapter le microscope électronique par transmission (MET) à émission de champ du CLYM (Centre Lyonnais de Microscopie) à la tomographie par rotation, (ii) ensuite, appliquer cette approche à l\u27étude de nanostructures hétérogènes ainsi que de nanomatériaux, (iii) finalement, explorer des méthodes 3D alternatives, comme la stéréoscopie, qui nécessite l\u27acquisition d\u27un nombre plus faible d\u27images qu\u27en tomographie électronique. Le travail expérimental a consisté à adapter l\u27embout du porte objet du MET, afin d\u27atteindre une plage de tilt au delà de 160 : une expérience de tomographie nécessite l\u27acquisition d\u27une centaine d\u27images sur différentes inclinaisons. Un logiciel a été développé pour contrôler semi-automatiquement le microscope et les conditions d\u27utilisation du détecteur, notamment la correction du focus dans les images durant la phase d\u27acquisition. Différents matériaux ont été étudiés: des nanoprécipités de carbure de vanadium (VC), des nanoparticules de catalyseurs (Pd), des nanocomposites de type "Au@SiOx" et un alliage présentant une nano-précipitation (AlZnMg)

Nanotomographie en mode STEM-HAAD

La tomographie électronique est une technique utilisée pour caractériser en 3D la structure et la chimie des matériaux, avec une résolution nanométrique dans le cas d'un microscope électronique par transmission. Le mode d'imagerie choisi est le champ sombre annulaire à grand angle, car il est adapté à la tomographie quantitative à la fois pour les échantillons cristallins et non-cristallins. De plus, le contraste en champ sombre annulaire dépend de la nature chimique des éléments observés, et la simulation des images permet d'extraire des informations chimiques, comme la densité volumique ou le numéro atomique des espèces chimiques présentes. L'objectif de cette thèse est triple : (i) dans un premier temps, adapter le microscope électronique par transmission (MET) à émission de champ du CLYM (Centre Lyonnais de Microscopie) à la tomographie par rotation, (ii) ensuite, appliquer cette approche à l'étude de nanostructures hétérogènes ainsi que de nanomatériaux, (iii) finalement, explorer des méthodes 3D alternatives, comme la stéréoscopie, qui nécessite l'acquisition d'un nombre plus faible d'images qu'en tomographie électronique. Le travail expérimental a consisté à adapter l'embout du porte objet du MET, afin d'atteindre une plage de tilt au delà de 160 : une expérience de tomographie nécessite l'acquisition d'une centaine d'images sur différentes inclinaisons. Un logiciel a été développé pour contrôler semi-automatiquement le microscope et les conditions d'utilisation du détecteur, notamment la correction du focus dans les images durant la phase d'acquisition. Différents matériaux ont été étudiés : des nanoprécipités de carbure de vanadium (VC), des nanoparticules de catalyseurs (Pd), des nanocomposites de type "Au@SiOx" et un alliage présentant une nano-précipitation (AlZnMg).Electron tomography is a technique used to characterise 3D structure and chemistry of the observed samples, with a nanometer resolution when applied in a Transmission Electron Microscope. The chosen imaging mode is STEM-HAADF (Scanning Transmission Electron Microscopy in the High Angle Annular Dark Field imaging mode) because it is well-adapted to a quantitative tomography, for both crystalline and amorphous materials. Moreover the STEM HAADF contrast is related to the chemical nature of elements, and simulation of images can be undertaken to extract chemical information, such as volume density or atomic number of particles. The aim of this thesis is threefold: (i) firstly to adapt the transmission electron microscope of the laboratory to tilting tomography, (ii) secondly to apply this approach to the study of heterogeneous nanostructures and nanomaterials, (iii) endly to explore alternative 3D methods, such as extended stereoscopy, which requires the acquisition of fewer images as compared to complete titlitng tomography . The experimental work has consisting in adaptating the tip of the TEM specimen holder in order to reach a tilt range up of 160, as a tomography experience requires acquisition of hundreds of images at different tilt. A software has been written to control semi-automatically the microscope and the detector, and especially to correct the focus in images during the phase of acquisition. The materials which have been studied are: nanoprecipitates of VC, Pd catalysts, Au@SiOx nanocomposites, and an AlZnMg alloyVILLEURBANNE-DOC'INSA LYON (692662301) / SudocSudocFranceF

3D-Geometrical and chemical quantification of Au@SiOx nano-composites in HAADF-STEM imaging mode

International audienceHybrid gold and Silica nano-composites (Au@SiOx) can be used for photonic, optical and biomedical applications, for example for the development of novel contrast agents in magnetic resonance imaging (e.g. [1]). According to the synthesis conditions, these nano-hybrids can adopt various configurations, such as core-shell structures, distribution of Au nano-particles exclusively inside, or at the surface of the silica-based particles. The synthesis involves micro-emulsions; the native micellar structure (oil in water, presence of surfactant, co-surfactant) defines the final silica morphology, and the localization of gold particles with respect to the silica ones depends on the order of adding nano-reactors. An optimised feedback on the synthesis conditions requires a detailed geometrical and chemical analysis of the final products. According to the nanometric size of these objects (see figure 1), a stereoscopy approach in HAADF (High Angle Annular Dark Field) imaging in a Transmission Electron Microscope appears to be an elegant way for that purpose

Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase

Eukaryotic vacuolar H+-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a central rotor subcomplex, with peripheral stalks preventing the entire membrane-bound region from turning with the rotor. The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions1,2. Studies of ATP synthases, V-ATPases, and bacterial/archaeal V/A-ATPases have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases3,4,5, but the structures of different rotational states have never been observed experimentally. Here we use electron cryomicroscopy to obtain structures for three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H+ ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane α-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.We thank Drs. P. Rosenthal, R. Henderson, V. Kanelis, and L. Kay for comments on the manuscript. JZ was supported by a Doctoral Postgraduate Scholarship from the National Science and Engineering Council of Canada and a Mary Gertrude l’Anson Scholarship. JLR is the Canada Research Chair in Electron Cryomicroscopy. This work was supported by operating grant MOP 81294 from the Canadian Institutes of Health Research

Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images

Single particle electron cryomicroscopy (cryo-EM) allows for structures of proteins and protein complexes to be determined from images of non-crystalline specimens. Cryo-EM data analysis requires electron microscope images of randomly oriented ice-embedded protein particles to be rotated and translated to allow for coherent averaging when calculating three-dimensional (3D) structures. Rotation of 2D images is usually done with the assumption that the magnification of the electron microscope is the same in all directions. However, due to electron optical aberrations, this condition is not met with some electron microscopes when used with the settings necessary for cryo-EM with a direct detector device (DDD) camera. Correction of images by linear interpolation in real space has allowed high-resolution structures to be calculated from cryo-EM images for symmetric particles. Here we describe and compare a simple real space method, a simple Fourier space method, and a somewhat more sophisticated Fourier space method to correct images for a measured anisotropy in magnification. Further, anisotropic magnification causes contrast transfer function (CTF) parameters estimated from image power spectra to have an apparent systematic astigmatism. To address this problem we develop an approach to adjust CTF parameters measured from distorted images so that they can be used with corrected images. The effect of anisotropic magnification on CTF parameters provides a simple way of detecting magnification anisotropy in cryo-EM datasets.We are grateful to Alexis Rohou and Timothy Grant from Nikolaus Grigorie 's laboratory, who quickly put us on the correct track when we noticed the apparent systematic astigmatism in plots of z1 versus z2. We thank Richard Henderson for critical comments on the manuscript. JZ was supported by a Doctoral Postgraduate Scholarship from the National Science and Engineering Council of Canada and a Mary Gertrude l'Anson Scholarship. JLR was supported by a Canada Research Chair. This work was funded by NSERC grant 401724-12 to JLR