Rasmus R. Schröder

Dimer ribbons of ATP synthase shape the inner mitochondrial membrane

ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo‐tomography we show that the ATP synthase of mammalian mitochondria is arranged in long ∼1‐μm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17‐nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of ∼0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton‐limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance.

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the “power stroke”, which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.

Fabrication of a Boersch phase plate for phase contrast imaging in a transmission electron microscope

The Boersch phase plate for a transmission electron microscope (TEM) offers major advantages over other phase plate concepts. However, due to its miniature dimensions, it could not be constructed and implemented so far. We report the first successful fabrication of a Boersch phase plate, which was produced by a combination of electron-beam and focused ion-beam lithography on a freestanding silicon nitride membrane. The manufactured multilayer electrode structure was tested for its functionality as an electrostatic einzel lens in a TEM. First experiments show that it can be used as a phase shifting device, as proposed by Boersch, to optimize phase contrast transfer in transmission electron microscopy.

Zero-loss energy filtering as improved imaging mode in cryoelectronmicroscopy of frozen-hydrated specimens☆

Abstract One of the remaining problems in attaining higher structural resolution with cryoelectronmicroscopy of frozen-hydrated specimens is the low contrast of micrographs taken close to the electron optical focus. By measuring electron energy loss spectra (EELS) of ice layers we show that a large fraction of incident electrons undergoes an inelastic electron-plasmon scattering process. Thus these electrons do not carry structural information of the protein but increase the background of the electron image and therefore reduce the contrast of the negative. Here we report the improvement in contrast gained by filtering out inelastically scattered electrons using an energy-filtered transmission electron microscope (EFTEM). This gain in contrast permits a dramatic decrease in defocusing values, resulting in improved structural resolution. In addition, the increased signal to noise ratio allows the recording of micrographs at a reduced electron dose. This should result in less damage to vitrified and unstained proteins and other beam-sensitive specimens.

Unraveling the nanoscale morphologies of mesoporous perovskite solar cells and their correlation to device performance.

Hybrid solar cells based on organometal halide perovskite absorbers have recently emerged as promising class for cost- and energy-efficient photovoltaics. So far, unraveling the morphology of the different materials within the nanostructured absorber layer has not been accomplished. Here, we present the first visualization of the mesoporous absorber layer in a perovskite solar cell from analytical transmission electron microscopy studies. Material contrast is achieved by electron spectroscopic imaging. We found that infiltration of the hole transport material into the scaffold is low and inhomogeneous. Furthermore, our data suggest that the device performance is strongly affected by the morphology of the TiO2 scaffold with a fine grained structure being disadvantageous.

Time−resolved cryo−electron microscopic study of the dissociation of actomyosin induced by photolysis of photolabile nucleotides

The rapid release of a substrate or other ligand from photolabile precursors in a thin layer suspension of biological specimens followed by rapid freezing provides a method of trapping and visualizing short-lived states in a dynamic system. We demonstrate here the first successful application of this method to study the interaction of actin filaments with myosin subfragment 1 (S1) after release of nucleotides. The results obtained suggest that structural changes in actin filaments occur as a result of interaction with S1.

Zero-loss image formation and modified contrast transfer theory in EFTEM

For a weak phase/weak amplitude object the information transfer in the imaging process of TEM is described by the common formalism of the contrast transfer function (CTF). So far the effects of inelastic scattering were not accounted for in this formalism. In conventional imaging they were simply neglected. In energy filtering TEM (EFTEM), where removal of inelastic electrons leads to higher specimen contrast, they were modelled by a global increase of the elastic amplitude contrast. Thus, the description of inelastic and elastic scattering was mixed. Here a new ansatz is proposed which treats elastic and inelastic contrast transfer separately by adding an inelastic contribution to the scattering potentials. In EFTEM this has the effect of adding a filter contrast which depends on the characteristics of the inelastic scattering. For samples with dominant plasmon loss the additional filter contrast is restricted to low resolution. Because of its strong dependence on the nature of the inelastic scattering process, the filter contrast cannot in general be unified with the conventional elastic amplitude contrast. The modified CTF theory for EFTEM was tested experimentally on a variety of samples. Images of amorphous layers of copper, aluminium, and carbon films, as well as zero-loss images of proteins embedded in amorphous ice were evaluated. The values of the parameters of the additional filter contrast were determined for carbon film and proteins embedded in vitrified ice. Comparison of different CTF models used to reconstruct 3D volumes from zero-loss images confirmed that best agreement with the atomic model is attained with the new, modified CTF theory.

Elastic and inelastic scattering cross-sections of amorphous layers of carbon and vitrified ice

Abstract Elastic and inelastic scattering cross-sections of amorphous layers of carbon and vitrified ice have been determined by electron spectroscopic diffraction (ESD). Using an energy-filtering TEM (EFTEM), elastic and inelastic differential scattering distributions were recorded separately on Image Plates (IP) and quantified. The thickness of carbon films was measured photometrically, that of ice layers by direct imaging. The elastic cross-sections are in good agreement with theory and previous experimental data. The measured inelastic scattering cross-sections are higher than the values derived from theoretical models for free atoms because these models do not account for collective excitations and binding effects. The short mean free path length for inelastic scattering indicates the importance of zero-loss energy filtering for imaging of biological samples embedded in amorphous ice.

Force-producing ADP state of myosin bound to actin.

Significance The force-generating state of myosin bound to actin is a key state in the chemomechanical cycle of this motor, but the structural details of the force-generating state are unknown. CryoEM structures of myosin V bound to actin with and without nucleotide reveal a previously unidentified conformation of elements associated with nucleotide binding and, most importantly, of the β-sheet that controls the position of nucleotide-binding elements. These specific structural features explain how myosin can bind strongly to both actin and MgADP, allowing force to be developed. Thus, this structure provides fundamental insights into how myosin works. It also provides insights into how strain may prevent release of MgADP, which is a critical property for the cellular roles of many myosins, including myosin V. Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.

Cryo-electron microscopic studies of relaxed striated muscle thick filaments

SummaryElectron micrograph images of rapidly frozen suspensions of thick filaments from four different muscle types are presented. Their optical and computer transforms are compared with images and diffraction patterns of negatively stained filaments and with X-ray data from the same muscles. We conclude that myosin head arrangement can be preserved on rapid freezing and that the images produced can be analysed by image processing techniques to give new information on thick filament structure.