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For cryo‐electron tomography (cryo‐ET) of beam‐sensitive biological specimens, a planar sample geometry is typically used. As the sample is tilted, the effective thickness of the sample along the direction of the electron beam increases and the signal‐to‐noise ratio concomitantly decreases, limiting the transfer of information at high tilt angles. In addition, the tilt range where data can be collected is limited by a combination of various sample‐environment constraints, including the limited space in the objective lens pole piece and the possible use of fixed conductive braids to cool the specimen. Consequently, most tilt series are limited to a maximum of ±70°, leading to the presence of a missing wedge in Fourier space. The acquisition of cryo‐ET data without a missing wedge, for example using a cylindrical sample geometry, is hence attractive for volumetric analysis of low‐symmetry structures such as organelles or vesicles, lysis events, pore formation or filaments for which the missing information cannot be compensated by averaging techniques. Irrespective of the geometry, electron‐beam damage to the specimen is an issue and the first images acquired will transfer more high‐resolution information than those acquired last. There is also an inherent trade‐off between higher sampling in Fourier space and avoiding beam damage to the sample. Finally, the necessity of using a sufficient electron fluence to align the tilt images means that this fluence needs to be fractionated across a small number of images; therefore, the order of data acquisition is also a factor to consider. Here, an n‐helix tilt scheme is described and simulated which uses overlapping and interleaved tilt series to maximize the use of a pillar geometry, allowing the entire pillar volume to be reconstructed as a single unit. Three related tilt schemes are also evaluated that extend the continuous and classic dose‐symmetric tilt schemes for cryo‐ET to pillar samples to enable the collection of isotropic information across all spatial frequencies. A fourfold dose‐symmetric scheme is proposed which provides a practical compromise between uniform information transfer and complexity of data acquisition. [ABSTRACT FROM AUTHOR]

The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 [beta]-polypeptides sandwiched between 17 [alpha]- and 16 [gamma]-polypeptides. Tight packing of the [gamma]-apoproteins between [beta]-polypeptides collectively interlocks and stabilizes the LH1 structure; this, together with the short Mg-Mg distances of bacteriochlorophyll b pairs, contributes to the large redshift of bacteriochlorophyll b absorption. The 'missing' 17th [gamma]-polypeptide creates a pore in the LH1 ring, and an adjacent binding pocket provides a folding template for a quinone, Q .sub.P, which adopts a compact, export-ready conformation before passage through the pore and eventual diffusion to the cytochrome bc.sub.1 complex. A cryo-electron microscopy structure of the light-harvesting-reaction centre (LH1-RC) complex of the photosynthetic bacterium Blastochloris viridis suggests factors that underlie the large redshift in the absorption spectrum of bacteriochlorophyll in the complex and that promote quinone-quinol translocation across the LH1 ring., Author(s): Pu Qian [sup.1] , C. Alistair Siebert [sup.2] , Peiyi Wang [sup.3] , Daniel P. Canniffe [sup.1] , C. Neil Hunter [sup.1] Author Affiliations: (1) Department of Molecular Biology [...]

Cytochrome bc1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b, cytochrome c1, and the Rieske iron–sulfur subunit, but the function of mitochondrial cytochrome bc1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc1 complex from the purple phototrophic bacterium Rhodobacter sphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene–maleic acid copolymer to purify the R. sphaeroides cytochrome bc1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c1 subunits. We observe a quinone at the Qo quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex. [ABSTRACT FROM AUTHOR]

In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ¡«10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn cofactor that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex. [ABSTRACT FROM AUTHOR]