Optimized cryo-EM data-acquisition workflow by sample-thickness determination

Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data-acquisition approaches in single-particle cryo-EM do not take it into account. Here, it is demonstrated how the sample thickness can be determined before data acquisition, allowing the identification of optimal regions and the restriction of automated data collection to images with preserved high-resolution details. This quality-over-quantity approach almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to a lack of high-resolution information. It maximizes the data-collection efficiency and lowers the electron-microscopy time required per data set. This strategy is especially useful if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck

The chlorosome: a prototype for efficient light harvesting in photosynthesis

Three phyla of bacteria include phototrophs that contain unique antenna systems, chlorosomes, as the principal light-harvesting apparatus. Chlorosomes are the largest known supramolecular antenna systems and contain hundreds of thousands of BChl c/d/e molecules enclosed by a single membrane leaflet and a baseplate. The BChl pigments are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Their excitation energy flows via a small protein, CsmA embedded in the baseplate to the photosynthetic reaction centres. Chlorosomes allow for photosynthesis at very low light intensities by ultra-rapid transfer of excitations to reaction centres and enable organisms with chlorosomes to live at extraordinarily low light intensities under which no other phototrophic organisms can grow. This article reviews several aspects of chlorosomes: the supramolecular and molecular organizations and the light-harvesting and spectroscopic properties. In addition, it provides some novel information about the organization of the baseplate

Fine structure of granal thylakoid membrane organization using cryo electron tomography

The architecture of grana membranes from spinach chloroplasts was studied by cryo electron tomography. Tomographic reconstructions of ice-embedded isolated grana stacks enabled to resolve features of photosystem II (PSII) in the native membrane and to assign the absolute orientation of individual membranes of granal thylakoid discs. Averaging of 3D sub-volumes containing PSII complexes provided a 3D structure of the PSII complex at 40 Å resolution. Comparison with a recently proposed pseudo-atomic model of the PSII supercomplex revealed the presence of unknown protein densities right on top of 4 light harvesting complex II (LHCII) trimers at the lumenal side of the membrane. The positions of individual dimeric PSII cores within an entire membrane layer indicates that about 23% supercomplexes must be of smaller size than full C2S2M2 supercomplexes, to avoid overlap.

Structural organization of the needle complex of the type III secretion apparatus of Shigella flexneri

The secretion apparatus known as the needle complex (NC) from the bacterium Shigella flexneri was studied by single particle electron microscopy. The isolated intact NC appears in projection to be composed of a basal body consisting of seven rings and a protruding needle appendage. A comparison of averaged projections of the intact NC and its fragments revealed the organization of the NC into several major subcomplexes. One of these lacks an inner membrane ring of the basal body but still presents the needle appendage attached to four upper rings. The position of the needle appendage within these rings is variable, suggesting that the dissociated component is necessary for stabilizing the needle appendage. Averaged images of the subcomplex lacking the inner membrane basal rings show a thicker extension at the base of the needle appendage, called the socket. This socket was also found to be present in images of the basal body fragment isolated from mutants lacking the mxiH and mxiI genes. This suggests that the socket is not composed of MxiH and MxiI subunits, which form the needle appendage. A symmetry analysis of the basal body top view projections indicated that a peripheral protein component of the inner membrane ring is present in a ring with 24 copies, in contrast to the Salmonella typhimurium NC. A model is presented in which the NC is only associated to the outer- and inner-membranes with its first and seventh ring, respectively.

Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca-bound and Ca-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement

Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+ binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement

Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography

The fine structure of intact, close-to-spherical mitochondria from the alga Polytomella was visualized by dual-axis cryo-electron tomography. The supramolecular organization of dimeric ATP synthase in the cristae membranes was investigated by averaging subvolumes of tomograms and 3D details at ~6 nm resolution were revealed. Oligomeric ATP synthase is composed of rows of dimers at 12 nm intervals; the dimers make a slight angle along the row. In addition, the main features of monomeric ATP synthase, such as the conically shaped F1 headpiece, central stalk and stator were revealed. This demonstrates the capability of dual-axis electron tomography to unravel details of proteins and their interactions in complete organelles.

Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+ -bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state

Chaplins of Streptomyces coelicolor self-assemble into two distinct functional amyloids

<p>Chaplins are small, secreted proteins of streptomycetes that play instrumental roles in the formation of aerial hyphae and attachment of hyphae to surfaces. Here we show that the purified proteins self-assemble at a water/air interface into an asymmetric and amphipathic protein membrane that has an amyloid nature. Cryo-tomography reveals that the hydrophilic surface is relatively smooth, while the hydrophobic side is highly structured and characterized by the presence of small fibrils, which are similar to those observed on the surfaces of aerial hyphae. Interestingly, our work also provides evidence that chaplins in solution assemble into amyloid fibrils with a distinct morphology. These hydrophilic fibrils strongly resemble the structures known to be involved in attachment of Streptomyces hyphae to surfaces. These data for the first time show the assembly of bacterial proteins into two distinct amyloid structures that have different and relevant functions in vivo. (C) 2013 Elsevier Inc. All rights reserved.</p>