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Electron cryo-microscopy of two-dimensional crystals of the major light-harvesting complex from pea chloroplasts has revealed the structure at 3A A resolution. The atomic model provides insights into the molecular architecture, function and assembly of this key component of the photosynthetic membrane and will enable us to understand how energy is transferred to the photosynthetic reaction centres.

Implementation of physical phase plates into transmission electron microscopes to achieve in-focus contrast for ice-embedded biological specimens poses several technological challenges. During the last decade several phase plates designs have been introduced and tested for electron ciyo-microscopy (cryoEM), including thin film (Zernike) phase plates and electrostatic devices. Boersch phase plates (BPPs) are electrostatic einzel lenses shifting the phase of the unscattered beam by an arbitrary angle. Adjusting the phase shift to 90 achieves the maximum contrast transfer for phase objects such as biomolecules. Recently, we reported the implementation of a BPP into a dedicated phase contrast aberration-corrected electron microscope (PACEM) and demonstrated its use to generate in-focus contrast of frozen-hydrated specimens. However, a number of obstacles need to be overcome before BPPs can be used routinely, mostly related to the phase plate devices themselves. CryoEM with a physical phase plate is affected by electrostatic charging, obliteration of low spatial frequencies, and mechanical drift. Furthermore, BPPs introduce single sideband contrast (SSB), due to the obstruction of Friedel mates in the diffraction pattern. In this study we address the technical obstacles in detail and show how they may be overcome. We use X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) to identify contaminants responsible for electrostatic charging, which occurs with most phase plates. We demonstrate that obstruction of low-resolution features is significantly reduced by lowering the acceleration voltage of the microscope. Finally, we present computational approaches to correct BPP images for SSB contrast and to compensate for mechanical drift of the BPP. (C) 2012 Elsevier B.V. All rights reserved.

Membrane proteins perform a wide range of essential functions in all cells of all living organisms, ranging from the sensing, processing and propagation of extrinsic signals, passive or active transport of ions and solutes, creating or utilizing a membrane potential, to the import or secretion of entire proteins. In spite of intense, and increasingly successful efforts in determining membrane protein structures, they still present a formidable challenge in structural biology.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F 1 heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

Anabaena sp. PCC 7120 is a prototype filamentous nitrogen-fixing cyanobacterium, in which nitrogen fixation and photosynthesis are spatially separated. Recent molecular and cellular studies have established the importance of molecular exchange between cells in the filament, but the routes involved are still under investigation. Two current models propose either a continuous periplasm or direct connections between adjacent cells whose integrity requires the protein SepJ. We used electron tomography to analyze the ultrastructure of the septum between vegetative cells in the Anabaena filament and were able to visualize intercellular connections that we term 'SEPTOSOMES'. We observed that, whereas the existence of the septosome does not depend on the presence of SepJ, the spacing between the two plasma membranes of the septum was significantly decreased in a ΔsepJ mutant. In addition, we observed that the peptidoglycan layer of each cell enters the septum but the outer membrane does not. Thus, each cell in the filament is individually surrounded by a plasma membrane and a peptidoglycan layer, and physical cell-cell contacts are mediated by the septosome.

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C(s) corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.

International audience; Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

Ultrathin carbon nanomembranes (CNM) comprising crosslinked biphenyl precursors have been tested as support films for energy-filtered transmission electron microscopy (EFTEM) of biological specimens. Due to their high transparency CNM are ideal substrates for electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) of stained and unstained biological samples. Virtually background-free elemental maps of tobacco mosaic virus (TMV) and ferritin have been obtained from samples supported by ~ 1 nm thin CNM. Furthermore, we have tested conductive carbon nanomembranes (cCNM) comprising nanocrystalline graphene, obtained by thermal treatment of CNM, as supports for cryoEM of ice-embedded biological samples. We imaged ice-embedded TMV on cCNM and compared the results with images of ice-embedded TMV on conventional carbon film (CC), thus analyzing the gain in contrast for TMV on cCNM in a quantitative manner. In addition we have developed a method for the preparation of vitrified specimens, suspended over the holes of a conventional holey carbon film, while backed by ultrathin cCNM.

For single particle electron cryo-microscopy (cryoEM), contrast loss due to beam-induced charging and specimen movement is a serious problem, as the thin films of vitreous ice spanning the holes of a holey carbon film are particularly susceptible to beam-induced movement. We demonstrate that the problem is at least partially solved by carbon nanotechnology. Doping ice-embedded samples with single-walled carbon nanotubes (SWNT) in aqueous suspension or adding nanocrystalline graphene supports, obtained by thermal conversion of cross-linked self-assembled biphenyl precursors, significantly reduces contrast loss in high-resolution cryoEM due to the excellent electrical and mechanical properties of SWNTs and graphene.

Two different thylakoid lipids are specifically associated with the light-harvesting complex of photosystem II (LHC-II). Digalactosyl diacyl glycerol (DGDG) binds to the isolated complex but can be removed by mild detergent treatment and anion-exchange chromatography. Removal of this lipid renders the complex unable to form two-dimensional or three-dimensional crystals. The ability to crystallize is completely restored by addition of pure DGDG, at a ratio of about four molecules per polypeptide for three dimensional crystals, suggesting several binding sites at the periphery of the trimeric complex. Two-dimensional crystals of purified protein grown in the presence of DGDG are more highly ordered than those obtained from the unfractionated complex. The other lipid, phosphatidyl glycerol (PG), binds more firmly and cannot be removed with non-ionic detergent. Complete delipidation of LHC-H can be achieved either with phospholipase or by proteolytic cleavage of 49 amino acid residues at the N terminus. Both treatments dissociate the native, trimeric complex into monomers. This indicates that PG is directly involved in the formation of trimers, which are a prerequisite for two dimensional and three-dimensional crystallization. Both lipids are therefore present in two dimensional and three-dimensional crystals and have distinct roles in the structure of the complex.

Precise knowledge of the structure of macromolecules in the cell is essential for understanding how they function. Structures of large macromolecules can now be obtained at near-atomic resolution by averaging thousands of electron microscope images recorded before radiation damage accumulates. This is what Amunts et al. have done in their research article on page 1485 of this issue ( 1 ), reporting the structure of the large subunit of the mitochondrial ribosome at 3.2 A resolution by electron cryo-microscopy (cryo-EM). Together with other recent high-resolution cryo-EM structures ( 2 – 4 ) (see the figure), this achievement heralds the beginning of a new era in molecular biology, where structures at near-atomic resolution are no longer the prerogative of x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy.

A high-resolution crystal structure of photosystem I, part of the machinery that performs photosynthesis, reveals how an extensive array of chlorophylls uses solar energy to transport electrons.

We applied dynamic single-molecule force spectroscopy to quantify the parameters (free energy of activation and distance of the transition state from the folded state) characterizing the energy barriers in the unfolding energy landscape of the outer membrane protein G (OmpG) from Escherichia coli. The pH-dependent functional switching of OmpG directs the protein along different regions on the unfolding energy landscape. The two functional states of OmpG take the same unfolding pathway during the sequential unfolding of β-hairpins I-IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a rigidified extracellular gating loop L6. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.

We have determined the structure of the archaeal sodium/proton antiporter NhaP1 at 7 Å resolution by electron crystallography of 2D crystals. NhaP1 is a dimer in the membrane, with 13 membrane-spanning α-helices per protomer, whereas the distantly related bacterial NhaA has 12. Dimer contacts in the two antiporters are very different, but the structure of a six-helix bundle at the tip of the protomer is conserved. The six-helix bundle of NhaA contains two partially unwound α-helices thought to harbour the ion-translocation site, which is thus similar in NhaP1. A model of NhaP1 based on detailed sequence comparison and the NhaA structure was fitted to the 7 Å map. The additional N-terminal helix 1 of NhaP1, which appears to be an uncleaved signal sequence, is located near the dimer interface. Similar sequences are present in many eukaryotic homologues of NhaP1, including NHE1. Although fully folded and able to dimerize, NhaP1 constructs without helix 1 are inactive. Possible reasons are investigated and discussed.

Cyclophilin D (CYPD) is a mitochondrial peptidyl prolyl-cis,trans-isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wild-type with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild-type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging.

The Na+-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 A in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 A, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.

The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the beta-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of beta-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore.

Single-molecule force spectroscopy (SMFS) is a unique approach to study the mechanical unfolding of proteins. Such forced unfolding experiments yield insight into how interactions stabilize a protein and guide its unfolding pathways. Previous SMFS work has probed the mechanical stability of water-soluble proteins composed of a helices and b strands. A prominent example of unfolding of a b-barrel structure is that of the green fluorescent protein (GFP), the stability of which plays a major role for its application as a marker in modern fluorescence microscopy. In contrast to the variety of water-soluble proteins characterized, only a-helical membrane proteins have been probed by SMFS. It was found that a-helical membrane proteins unfold via many intermediates, which is different to the mostly two-state unfolding process of water-soluble proteins. Upon mechanically pulling the peptide end of a membrane protein, single and grouped a helices and polypeptide loops unfold in steps until the entire protein has unfolded. Whether the a helices and loops unfold individually or cooperatively to form an unfolding intermediate depends on the interactions established within the membrane protein and with the environment. Each of these unfolding events creates an unfolding intermediate with the sequence of intermediates describing the unfolding pathway taken. However, so far, b-barrel-forming membrane proteins have not been characterized by SMFS. For these reasons, we have characterized the interactions and unfolding of the b-barrel-forming outer-membrane protein OmpG from Escherichia coli by SMFS. The structure of OmpG comprises 14 b strands that form a transmembrane b-barrel pore. Six short loops (T1–T6) on the periplasmic side and seven longer loops (L1–L7) on the extracellular side connect the individual b strands. OmpG is gated by loop L6, which controls the flux of small molecules through the pore and the permeability of the bacterial outer membrane in a pH-dependent manner. Being able to withstand rather harsh environmental conditions, OmpG forms a robust pore, which makes it suitable for application as a biosensor. In our SMFS experiments, OmpG reconstituted in E. coli lipid membranes were first imaged by AFM. The AFM tip was then pushed onto the OmpG surface to facilitate the nonspecific attachment of the N or C terminus (Figure 1a).

FeoB in bacteria and archaea is involved in the uptake of ferrous iron (Fe2+), an important cofactor in biological electron transfer and catalysis. Unlike any other known prokaryotic membrane protein, FeoB contains a GTP-binding domain at its N-terminus. We determined high-resolution X-ray structures of the FeoB G-domain from Methanococcus jannaschii with and without bound GDP or Mg2+-GppNHp. The G-domain forms the same dimer in all three structures, with the nucleotide-binding pockets at the dimer interface, as in the ATP-binding domain of ABC transporters. The G-domain follows the typical fold of nucleotide-binding proteins, with a β-strand inserted in switch I that becomes partially disordered upon GTP binding. Switch II does not contact the nucleotide directly and does not change its conformation in response to the bound nucleotide. Release of the nucleotide causes a rearrangement of loop L6, which we identified as the G5 region of FeoB. Together with the C-terminal helix, this loop may transmit the information about the nucleotide-bound state from the G-domain to the transmembrane region of FeoB.

Electron diffraction patterns of two-dimensional crystals of light-harvesting chlorophyll a/b-protein complex (LHC-II) from photosynthetic membranes of pea chloroplasts, tilted at different angles up to 60 degrees , were collected to 3.2 A resolution at -125 degrees C. The reflection intensities were merged into a three-dimensional data set. The Friedel R-factor and the merging R-factor were 21.8 and 27.6%, respectively. Specimen flatness and crystal size were critical for recording electron diffraction patterns from crystals at high tilts. The principal sources of experimental error were attributed to limitations of the number of unit cells contributing to an electron diffraction pattern, and to the critical electron dose. The distribution of strong diffraction spots indicated that the three-dimensional structure of LHC-II is less regular than that of other known membrane proteins and is not dominated by a particular feature of secondary structure.

The chlorophyll a/b light-harvesting complex of photosystem II (LHC-II) collects most of the solar energy in the biosphere. LHC-II is the prototype of a highly conserved family of membrane proteins that fuels plant photosynthesis in the conversion of excitation energy into biologically useful chemical energy. In addition, LHC-II plays an important role in the organisation of the thylakoid membrane, the structure of the photosynthetic apparatus, the regulation of energy flow between the two photosystems, and in the controlled dissipation of excess excitation energy under light stress. Our current understanding of the sophisticated mechanisms behind each of these processes has profited greatly from the progress made over the past two decades in determining the structure of the complex. This review presents the developments and breakthroughs that ultimately lead to the high-resolution structure of LHC-II. Based on an alignment of the remarkably well engineered and highly conserved LHC polypeptide, we propose several key features of the LHC-II structure that are likely to be present in all members of the LHC family. Finally, some recently proposed mechanisms of energy-dependent non-photochemical quenching (NPQ) are examined from a structural perspective.

Plants dissipate excess excitation energy as heat by non-photochemical quenching (NPQ). NPQ has been thought to resemble in vitro aggregation quenching of the major antenna complex, light harvesting complex of photosystem II (LHC-II). Both processes are widely believed to involve a conformational change that creates a quenching centre of two neighbouring pigments within the complex. Using recombinant LHC-II lacking the pigments implicated in quenching, we show that they have no particular role. Single crystals of LHC-II emit strong, orientation-dependent fluorescence with an emission maximum at 680 nm. The average lifetime of the main 680 nm crystal emission at 100 K is 1.31 ns, but only 0.39 ns for LHC-II aggregates under identical conditions. The strong emission and comparatively long fluorescence lifetimes of single LHC-II crystals indicate that the complex is unquenched, and that therefore the crystal structure shows the active, energy-transmitting state of LHC-II. We conclude that quenching of excitation energy in the light-harvesting antenna is due to the molecular interaction with external pigments in vitro or other pigment–protein complexes such as PsbS in vivo, and does not require a conformational change within the complex.

Recent theoretical and experimental studies reveal the central role of zeaxanthin radical cations (Zea•+) in regulation of photosynthetic light harvesting. Two-color two-photon spectroscopy on LHC-II protein CP29 reveals the in-situ photodynamics of Zea•+.

The surface roughness of carbon films, which are used as a specimen support in high-resolution electron microscopy, has been investigated by atomic force microscopy. Carbon films were prepared by evaporating carbon onto mica. We found that the carbon surface that had been in contact with the mica was between 3 and 9 times smoother than the surface which had faced the carbon source. Surfaces of carbon films prepared by multiple evaporation were smoother than films evaporated in a single step. The surface roughness on the mica side of these films was close to that of mica. Two-dimensional crystals of plant light-harvesting complex yielded isotropically sharp high-resolution electron diffraction patterns at high tilt angles only when supported on the smoothest carbon films, produced by multiple evaporation.

NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H+ and Na+. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na+ and Li+ at pH above 7 and involves a 7-A displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for proteins imported into mitochondria. We determined the structure of the native, unstained approximately 550-kDa core-Tom20 complex from Saccharomycescerevisiae by cryo-electron microscopy at 18-A resolution. The complex is triangular, measuring 145 A on edge, and has near-3-fold symmetry. Its bulk is made up of three globular approximately 50-A domains. Three elliptical pores on the c-face merge into one central approximately 70-A cavity with a cage-like assembly on the opposite t-face. Nitrilotriacetic acid-gold labeling indicates that three Tom22 subunits in the TOM complex are located at the perimeter of the complex near the interface of the globular domains. We assign Tom22, which controls complex assembly, to three peripheral protrusions on the c-face, while the Tom20 subunit is tentatively assigned to the central protrusion on this surface. Based on our three-dimensional map, we propose a model of transient interactions and functional dynamics of the TOM assembly.

Thin films of the metal glass Ti88Si12 were produced by evaporation and characterized by AFM and conductivity measurements. Thin Ti88Si12 support films for electron microscopy were prepared by coating standard EM grids with evaporated films floated off mica, and characterized by electron imaging and electron diffraction. At room temperature, the specific resistance of a thin TiSi film was 10(6) times lower than that of an amorphous carbon film. At 77K, the specific resistance of TiSi films decreased, whereas that of carbon became immeasurably high. The effective scattering cross-section of TiSi and amorphous carbon for 120 kV electrons is roughly equal, but TiSi films for routine use can be approximately 10 times thinner due to their high mechanical strength, so that they would contribute less background noise to the image. Electron diffraction of purple membrane on a TiSi substrate confirmed that the support film was amorphous, and indicated that the high-resolution order of the biological sample was preserved. Electron micrographs of TiSi films tilted by 45 degrees relative to the electron beam recorded at approximately 4 K indicated that the incidence of beam-induced movements was reduced by 50% compared to amorphous carbon film under the same conditions. The success rate of recording high-resolution images of purple membranes on TiSi films was close to 100%. We conclude that TiSi support films are ideal for high-resolution electron cryo-microscopy (cryo-EM) of biological specimens, as they reduce beam-induced movement significantly, due to their high electrical conductivity at low temperature and their favorable mechanical properties.

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. Recently, carotenoid radical cation (Car*+) formation has been discovered to be a key step for the feedback deexcitation quenching mechanism (qE), a component of NPQ, of which the molecular mechanism and location is still unknown. We have generated and characterized carotenoid radical cations by means of resonant two color, two photon ionization (R2C2PI) spectroscopy. The Car*+ bands have maxima located at 830 nm (violaxanthin), 880 nm (lutein), 900 nm (zeaxanthin), and 920 nm (beta-carotene). The positions of these maxima depend strongly on solution conditions, the number of conjugated C=C bonds, and molecular structure. Furthermore, R2C2PI measurements on the light-harvesting complex of photosystem II (LHC II) samples with or without zeaxanthin (Zea) reveal the violaxanthin (Vio) radical cation (Vio*+) band at 909 nm and the Zea*+ band at 983 nm. The replacement of Vio by Zea in the light-harvesting complex II (LHC II) has no influence on the Chl excitation lifetime, and by exciting the Chls lowest excited state, no additional rise and decay corresponding to the Car*+ signal observed previously during qE was detected in the spectral range investigated (800-1050 nm). On the basis of our findings, the mechanism of qE involving the simple replacement of Vio with Zea in LHC II needs to be reconsidered.

Although nonphotochemical quenching (NPQ) has been documented for years, its mechanism and exact location is still under debate. Femtosecond spectroscopy on LHCII and thylakoid membranes reveal that zeaxanthin (Zea) bound to the violaxanthin (Vio) site in isolated LHCII is not sufficient for efficient quenching.

The glutamate transporters GltP(Ec) from Escherichia coli and GltP(Ph) from Pyrococcus horikoshii were overexpressed in E. coli and purified to homogeneity with a yield of 1-2 mg/L of culture. Single-particle analysis and electron microscopy indicate that GltP(Ph) is a trimer in detergent solution. Electron microscopy of negatively stained GltP(Ph) two-dimensional crystals shows that the transporter is a trimer also in the membrane. Gel filtration of GltP(Ec) indicates a reversible equilibrium of two oligomeric states in detergent solution that we identified as a trimer and hexamer by blue-native gel electrophoresis and cross-linking. The purified transporters were fully active upon reconstitution into liposomes, as demonstrated by the uptake of radioactively labeled L-aspartate or L-glutamate. L-aspartate/L-glutamate transport of GltP(Ec) involves the cotransport of protons and depends only on pH, whereas GltP(Ph) catalyzes L-glutamate transport with a cotransport of H+ or Na+. L-glutamate induces a fast transient current in GltP(Ph) proteoliposomes coupled to a solid supported membrane (SSM). We show that the electric signal depends on the concentration of Na+ or H+ outside the proteoliposomes and that GltP(Ph) does not require K+ inside the proteoliposomes. In addition, the electrical currents are inhibited by TBOA and HIP-B. The half-saturation concentration for activation of GltP(Ph) glutamate transport (K0.5(glut)) is 194 microM.

Planar-tubular two-dimensional (2D) crystals of human vitamin K-dependent gamma-glutamyl carboxylase grow in the presence of dimyristoyl phosphatidylcholine (DMPC). Surprisingly, these crystals form below the phase transition temperature of DMPC and at the unusually low molar lipid-to-protein (LPR) ratio of 1, while 2D crystals are conventionally grown above the phase transition temperature of the reconstituting lipid and significantly higher LPRs. The crystals are up to 0.75 microm in the shorter dimension of the planar tubes and at least 1 microm in length. Due to the planar-tubular nature of the crystals, two lattices are present. These are rotated by nearly 90 degrees in respect to each other. The ordered arrays exhibit p12(1) plane group symmetry with unit cell dimensions of a=83.7 A, b=76.6 A, gamma=91 degrees. Projection maps calculated from images of negatively stained and electron cryo-microscopy samples reveal the human vitamin K-dependent gamma-glutamyl carboxylase to be a monomer.

Mechanisms of folding and misfolding of membrane proteins are of interest in cell biology. Recently, we have established single-molecule force spectroscopy to observe directly the stepwise folding of the Na+/H+ antiporter NhaA from Escherichia coli in vitro. Here, we improved this approach significantly to track the folding intermediates of a single NhaA polypeptide forming structural segments such as the Na+-binding site, transmembrane alpha-helices, and helical pairs. The folding rates of structural segments ranged from 0.31 s(-1) to 47 s(-1), providing detailed insight into a distinct folding hierarchy of an unfolded polypeptide into the native membrane protein structure. In some cases, however, the folding chain formed stable and kinetically trapped non-native structures, which could be assigned to misfolding events of the antiporter.

The glutamate transporter GLT-1 from Rattus norvegicus was expressed at high level in BHK cells using the Semliki Forest virus expression system. BHK cells infected with viral particles carrying the GLT-1 gene exhibited 30-fold increased aspartate uptake compared to control cells. The expression level of GLT-1 as determined by binding of labelled substrate to membrane preparations was about 3.5×10 6 functional transporters per cell, or 61 pmol GLT-1 per milligram of membrane protein. Purification of the His-tagged protein by Ni 2+ -NTA affinity chromatography enabled the routine production and purification of milligram quantities of fully functional transporter. Transport activity required reducing conditions and the addition of extra lipid throughout the purification. The apparent molecular mass of the recombinant transporter was 73 kDa or 55 kDa, corresponding to the glycosylated and non-glycosylated form, respectively. Both forms were active upon separation on a lectin column and reconstitution into liposomes. Glycosylated and non-glycosylated GLT-1 were transported to the plasma membrane with equal efficiency. Our results show that N-glycosylation does not affect the trafficking or the transport activity of GLT-1. The low-resolution structure of GLT-1 was determined by electron microscopy and single particle reconstruction.

Leukotriene (LT) C 4 synthase, an 18 kDa integral membrane enzyme, conjugates LTA 4 with reduced glutathione to form LTC 4 , the parent compound of all cysteinyl leukotrienes that play a crucial role in the pathobiology of bronchial asthma. We have calculated a projection map of recombinant human LTC 4 synthase at a resolution of 4.5 A by electron crystallography, which shows that the enzyme is a trimer. A map truncated at 7.5 A visualizes four transmembrane α helices per protein monomer. The densities in projection indicate that most of the α helices run nearly perpendicular to the plane of the membrane. At this resolution, LTC 4 synthase is strikingly similar to microsomal glutathione S-transferase 1, which belongs to the same gene family but bears little sequence identity and no resemblance in substrate specificity to the LTC 4 synthase. These results provide new insight into the structure and function of membrane proteins involved in eicosanoid and glutathione metabolism.

A detailed comparison of key components in the Photosystem II complexes of higher plants and cyanobacteria was carried out. While the two complexes are overall very similar, significant differences exist in the relative orientation of individual components relative to one another. We compared a three-dimensional map of the inner part of plant PS II at 8 A resolution, and a 5.5 A projection map of the same complex determined by electron crystallography, to the recent 3.5-3.8 A X-ray structures of cyanobacterial complexes. The largest differences were found in the rotational alignment of the cyt b(;)559 subcomplex, and of the CP47 core antenna with respect to the D1/D2 reaction centre. Within the D1/D2 proteins, there are clear differences between plants and cyanobacteria at the stromal ends of membrane-spanning helices, even though these proteins are highly homologous. Notwithstanding these differences in the protein scaffold, the distances between the critical photosynthetic pigment cofactors seem to be precisely conserved. The different protein arrangements in the two complexes may reflect an adaptation to the two very different antenna systems, membrane-extrinsic phycobilisomes for cyanobacteria, and membrane-embedded chlorophyll a/b proteins in plants.

P-type ATPases are ion pumps that carry out many fundamental processes in biology and medicine, ranging from the generation of membrane potential to muscle contraction and the removal of toxic ions from cells. Making use of the energy stored in ATP, they transport specific ions across the cell membrane against a concentration gradient. Recent X-ray structures and homology models of P-type pumps now provide a basis for understanding the molecular mechanism of ATP-driven ion transport.

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.

The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum , was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in β-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E. coli polar lipids produced 2D crystals with unit cell parameters of 182 A×154 A, γ=90° exhibiting p 22 1 2 1 symmetry. Electron cryo-microscopy yielded a projection map at 7.5 A. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane α-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.

The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Deltapsi as the only external energy source. We purified the protein insertion complex (TIM22 complex), a twin-pore translocase that mediated the insertion of precursor proteins in a three-step process. After the precursor is tethered to the translocase without losing energy from the Deltapsi, two energy-requiring steps were needed. First, Deltapsi acted on the precursor protein and promoted its docking in the translocase complex. Then, Deltapsi and an internal signal peptide together induced rapid gating transitions in one pore and closing of the other pore and drove membrane insertion to completion. Thus, protein insertion was driven by the coordinated action of a twin-pore complex in two voltage-dependent steps.

Membrane proteins still present a major challenge in structural biology. Electron microscopy (EM) of 2D crystals is an increasingly important technique for studying their structure and function. While the basic techniques and concepts are unchanged and valid, the number of membrane proteins crystallized in 2D has risen encouragingly in the past three or four years, no doubt largely due to the recent progress in expressing membrane proteins in homologous or heterologous systems. The evaluation of more than 60 2D crystallization protocols now provides a sound base for some general guidelines. This chapter discusses recent technical developments, which may help to facilitate the future production of 2D crystals. Several other reviews published in the past 10 years address various aspects of 2D crystallisation.

Mitochondria Cristae Karen M. Davies, Bertram Daum, Claudio Anselmi, Jose D. Faraldo-Gomez, Werner Kuhlbrandt Max Planck Institute of Biophysics, Department of Structural Biology, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany. Max Planck Institute of Biophysics, Department of Theoretical Molecular Biophysics Group and Cluster of Excellence ‘Macromolecular Complexes’, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany.

The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself.

Proton pumps in the plasma membrane of plants and yeasts maintain the intracellular pH and membrane potential. To gain insight into the molecular mechanisms of proton pumping, we built an atomic homology model of the proton pump based on the 2.6 angstrom x-ray structure of the related Ca 2+ pump from rabbit sarcoplasmic reticulum. The model, when fitted to an 8 angstrom map of the Neurospora proton pump determined by electron microscopy, reveals the likely path of the proton through the membrane and shows that the nucleotide-binding domain rotates by ∼70° to deliver adenosine triphosphate (ATP) to the phosphorylation site. A synthetic peptide corresponding to the carboxyl-terminal regulatory domain stimulates ATPase activity, suggesting a mechanism for proton transport regulation.

International audience; Transport and membrane integration of polypeptides is carried out by specific protein complexes in the membranes of all living cells. The Sec transport path provides an essential and ubiquitous route for protein translocation. In the bacterial cytoplasmic membrane, the channel is formed by oligomers of a heterotrimeric membrane protein complex consisting of subunits SecY, SecE and SecG. In the endoplasmic reticulum membrane, the channel is formed from the related Sec61 complex. Here we report the structure of the Escherichia coli SecYEG assembly at an in-plane resolution of 8 A. The three-dimensional map, calculated from two-dimensional SecYEG crystals, reveals a sandwich of two membranes interacting through the extensive cytoplasmic domains. Each membrane is composed of dimers of SecYEG. The monomeric complex contains 15 transmembrane helices. In the centre of the dimer we observe a 16 x 25 A cavity closed on the periplasmic side by two highly tilted transmembrane helices. This may represent the closed state of the protein-conducting channel.