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Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochrome b6/f complex are discussed.

Chlorosomes are sac-like, light-harvesting organelles that characteristically contain very large numbers of bacteriochlorophyll (BChl) c, d, or e molecules. These antenna structures occur in chlorophototrophs belonging to some members of the Chlorobi and Chloroflexi phyla and are also found in a recently discovered member of the phylum Acidobacteria, "Candidatus Chloracidobacterium thermophilum." "Ca. Chloracidobacterium thermophilum" is the first aerobic organism discovered to possess chlorosomes as light-harvesting antennae for phototrophic growth. Chlorosomes were isolated from "Ca. Chloracidobacterium thermophilum" and subjected to electron microscopic, spectroscopic, and biochemical analyses. The chlorosomes of "Ca. Chloracidobacterium thermophilum" had an average size of similar to 100 by 30 nm. Cryo-electron microscopy showed that the BChl c molecules formed folded or twisted, sheet-like structures with a lamellar spacing of similar to 2.3 nm. Unlike the BChls in the chlorosomes of the green sulfur bacterium Chlorobaculum tepidum, concentric cylindrical nanotubes were not observed. Chlorosomes of "Ca. Chloracidobacterium thermophilum" contained a homolog of CsmA, the BChl a-binding, baseplate protein; CsmV, a protein distantly related to CsmI, CsmJ, and CsmX of C. tepidum, which probably binds a single [2Fe-2S] cluster; and five unique polypeptides (CsmR, CsmS, CsmT, CsmU, and a type II NADH dehydrogenase homolog). Although "Ca. Chloracidobacterium thermophilum" is an aerobe, energy transfer among the BChls in these chlorosomes was very strongly quenched in the presence of oxygen (as measured by quenching of fluorescence emission). The combined analyses showed that the chlorosomes of "Ca. Chloracidobacterium thermophilum" possess a number of unique features but also share some properties with the chlorosomes found in anaerobic members of other phyla.

The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III 2 , and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III 2 and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.

A competitive photoresponsive supramolecular system is formed in a dilute aqueous solution of three components: vesicles of amphiphilic alpha-cyclodextrin host 1a, divalent p-methylphenyl guest 2 or divalent p-methylbenzamide guest 3, and photoresponsive azobenzene monovalent guest 5. Guests 2 and 3 form weak inclusion complexes with 1a (K(a)approximate to 10(2) M(-1)), whereas azobenzene guest 5 forms a strong inclusion complex (K(a)approximate to 10(4) M(-1)), provided it is in the trans state. The aggregation and adhesion of vesicles of host 1a is mediated by guest 2 (or 3) due to the formation of multiple intervesicular noncovalent links, as confirmed by using isothermal titration calorimetry (ITC), optical density measurements at 600 nm (OD600), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). The addition of excess monovalent guest trans-5 to vesicles of 1a aggregated by divalent guest 2 (or 3) causes the dispersion of vesicles of 1a because trans-5 displaces 2 (as well as 3) from the vesicle surface. Upon UV irradiation of a dilute ternary mixture of vesicles of 1a, guest 2 (or 3), and competitor trans-5, compound trans-5 isomerizes to cis-5, and renewed aggregation of vesicles of 1a by guest 2 (or 3) occurs because 2 (as well as 3) displaces cis-5 from the vesicle surface. Subsequent visible irradiation causes the redispersion of vesicles of 1a because cis-5 reisomerizes into trans-5, which again displaces guest 2 (or 3) from the vesicle surface. In this way, the competitive photoresponsive aggregation and dispersion of vesicles can be repeated for several cycles.

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated ( cas ) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a “seed sequence” at the 5′ end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli , termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.

PsbW, a 6.1-kDa low-molecular-weight protein, is exclusive to photosynthetic eukaryotes, and associates with the photosystem II (PSII) protein complex. In vivo and in vitro comparison of Arabidopsis thaliana wild-type plants with T-DNA insertion knock-out mutants completely lacking the PsbW protein, or with antisense inhibition plants exhibiting decreased levels of PsbW, demonstrated that the loss of PsbW destabilizes the supramolecular organization of PSII. No PSII-LHCII supercomplexes could be detected or isolated in the absence of the PsbW protein. These changes in macro-organization were accompanied by a minor decrease in the chlorophyll fluorescence parameter F(V) /F(M) , a strongly decreased PSII core protein phosphorylation and a modification of the redox state of the plastoquinone (PQ) pool in dark-adapted leaves. In addition, the absence of PsbW protein led to faster redox changes in the PQ pool, i.e. transitions from state 1 to state 2, as measured by changes in stationary fluorescence (F(S) ) kinetics, compared with the wild type. Despite these dramatic effects on macromolecular structure, the transgenic plants exhibited no significant phenotype under normal growth conditions. We suggest that the PsbW protein is located close to the minor antenna of the PSII complex, and is important for the contact and stability between several PSII-LHCII supercomplexes.

In this work we have purified the Photosystem I (PSI) complex of Chlamydomonas reinhardtii to homogeneity. Biochemical, proteomic, spectroscopic, and structural analyses reveal the main properties of this PSI-LHCI supercomplex. The data show that the largest purified complex is composed of one core complex and nine Lhca antennas and that it contains all Lhca gene products. A projection map at 15 Å resolution obtained by electron microscopy reveals that the Lhcas are organized on one side of the core in a double half-ring arrangement, in contrast with previous suggestions. A series of stable disassembled PSI-LHCI intermediates was purified. The analysis of these complexes suggests the sequence of the assembly/disassembly process. It is shown that PSI-LHCI of C. reinhardtii is larger but far less stable than the complex from higher plants. Lhca2 and Lhca9 (the red-most antenna complexes), although present in the largest complex in 1:1 ratio with the core, are only loosely associated with it. This can explain the large variation in antenna composition of PSI-LHCI from C. reinhardtii found in the literature. The analysis of several subcomplexes with reduced antenna size allows determination of the position of Lhca2 and Lhca9 and leads to a proposal for a model of the organization of the Lhcas within the PSI-LHCI supercomplex. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

A potentially scalable self-assembly method for single-walled carbon nanotubes (SWNTs) involves the use of amphiphilic DNA block copolymers. One such hybrid is able to cover the entire area of solution-based SWNT technologies, from selective dispersion to nondestructive functionalization to high-yield device fabrication such as field-effect transistors.

Photosystem I particles from Prochlorothrix hollandica were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose gradients in the presence of dodecylmaltoside. The particles were characterized by electron microscopy and image reconstruction techniques. The lowest, major green band in the gradient contained trimeric Photosystem I particles with features similar to Photosystem I from cyanobacteria. No evidence for an association of the chlorophyll a/b-binding antenna with Photosystem I was found. The trimeric association of Photosystem I appears to be a generally occurring aggregation mechanism in oxygenic prokaryotes.

The structure and aggregation behavior of B800-850 (LHII) and B875 (LHI) antenna complexes of Rhodobacter sphaeroides were studied by electron microscopy. Single molecular projections (top views and side views) of isolated particles were analyzed. The B800-850 complexes, isolated as 150 kDa particles, are cylindrical with a diameter of 8.5 nm and with a height of 6.5 nm. If corrected for attached detergent, the actual diameter in the plane of the membrane would be about 5.1 nm. Stain accumulation in the center of the structure indicates a small indentation, therefore a ring-shaped structure is expected. The size, shape and estimated mass give evidence for a B800-850 complex structure consisting of 4-6 alphabeta heterodimers, if in an alpha(n)beta(n) ring-shaped configuration. The B875 complexes, isolated as 360 kDa particles, are dimeric units, but the two monomers are only loosely connected. A single B875 unit has a corrected diameter of about 5.2 nm and a height of 6.2 nm, similar to the B800-850 unit. Therefore, the B875 complex most likely has the same alpha(n)beta(n) configuration. Image analysis of B800-850 projections points to a 3-or 6-fold symmetry in the top views, rather than a 4- or 5-fold symmetry. Therefore, our results are most compatible with an alpha6beta6 configuration.

Structural differences between oxy-hemocyanin and deoxy-hemocyanin from the spiny lobster P. interruptus were studied by electron microscopy and image analysis of negatively stained preparations. Projections of the hexameric P. interruptus hemocyanin from electron microscopy were compared with simulated projections of the high-resolution structure determined by X-ray crystallography previously. Image analysis was focused on the hexagonal views. Four independent data sets of hexagons (two of oxy- and two of deoxy-preparations) were analyzed and compared. Despite the small size of the projections, a resolution in the average images of better than 1.5 nm was obtained. A significant difference in stain modulation could be noticed between the oxy- and deoxy-preparations in two areas of the projections. The first difference in stain exclusion occurred in the center of the hexagonal views. The second is the shape of the domain 3 region. In the deoxy-images this region has a triangular shape, in the oxy-images it appears to be more rounded. The overall differences suggest a “breathing” of the hexamer upon oxygenation/deoxygenation.

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 angstrom 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. (C) 2010 Elsevier B.V. All rights reserved.

Tetrahymena ATP synthase, an evolutionarily divergent protein complex, has a very unusual structure and protein composition including a unique Fo subunit a and at least 13 proteins with no orthologs outside of the ciliate lineage., The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F1 sector catalyzes ATP synthesis, whereas the Fo sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F1 and Fo sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the Fo sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the Fo sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics., Author Summary Synthesis of ATP, the currency of the cellular energy economy, is carried out by a rotary nano-motor, the ATP synthase complex, which uses proton flow to drive the rotation of protein subunits so as to produce ATP. There are two main components in mitochondrial F-type ATP synthase complexes, each made up of a number of different proteins: F1 has the catalytic sites for ATP synthesis, and Fo forms channels for proton movement and provides a bearing and stator to contain the rotary action of the motor. The two parts of the complex have to interact with each other, and critical protein subunits of the enzyme are conserved from bacteria to higher eukaryotes. We were surprised that a group of unicellular organisms called alveolates (including ciliates, apicomplexa, and dinoflagellates) seemed to lack two critical proteins of the Fo component. We have isolated intact ATP synthase complexes from the ciliate Tetrahymena thermophila and examined their structure by electron microscopy and their protein composition by mass spectrometry. We found that the ATP synthase complex of this organism is quite different, both in its overall structure and in many of the associated protein subunits, from the ATP synthase in other organisms. At least 13 novel proteins are present within this complex that have no orthologs in any organism outside of the ciliates. Our results suggest significant divergence of a critical bioenergetic player within the alveolate group.

Cyanobacterial NDH-1 is a multisubunit complex involved in proton translocation, cyclic electron flow around photosystem I and CO(2) uptake. The function and location of several of its small subunits are unknown. In this work, the location of the small subunits NdhL, -M, -N, -O and CupS of Synechocystis 6803 NDH-1 was established by electron microscopy (EM) and single particle analysis. To perform this, the subunits were enlarged by fusion with the YFP protein. After classification of projections, the position of the YFP tag was revealed; all five subunits are integrated in the membrane domain. The results on NDH-1 demonstrate that a GFP tag can be revealed after data processing of EM data sets of moderate size, thus showing that this way of labeling is a fast and reliable way for subunit mapping in multisubunit complexes after partial purification. (C) 2010 Elsevier B.V. All rights reserved.

The five complexes (complexes I-V) of the oxidative phosphorylation (OXPHOS) system of mitochondria can be extracted in the form of active supercomplexes. Single-particle electron microscopy has provided 2D and 3D data describing the interaction between complexes I and III, among I. Ill and IV and in a dimeric form of complex V. between two ATP synthase monomers. The stable interactions are called supercomplexes which also form higher-ordered oligomers. Cryo-electron tomography provides new insights on how these supercomplexes are arranged within intact mitochondria. The structure and function of OXPHOS supercomplexes are discussed. (C) 2009 Elsevier B.V. All rights reserved.

The structure of the Photosystem I (PS I) complex from the thermophilic cyanobacterium Synechococcus sp. has been investigated by electron microscopy and image analysis of two-dimensional crystals. Crystals were obtained from isolated PS I by removal of detergents with Bio-Beads. After negative staining, either single layers or two superimposed layers with a rotational different orientation were observed. The layers have a rectangular unit cell of 16.0 x 15.0 nm, which contains two PS I monomers. The monomers are arranged alternating up and down in each layer. For double-layer crystals, the images of the two layers could be separately processed by a combination of Fourier-peak-filtering and correlation averaging. Features in the two-dimensional plane can be seen with a resolution up to 1.5-1.8 nm. A model for the PS I structure was obtained by combining three-dimensional reconstructions from three tilt-series. The model shows an asymmetric PS I complex. On one side (presumably the stromal side) there is a 3 nm high ridge. This is most likely comprised of the psaC, psaD and psaE subunits. The other side (presumably the lumenal side) is rather flat, but in the center there is a 3 nm deep indentation, which possibly separates partly the two large subunits psaA and psaB.

The molecular structure of groEL-like protein from pea leaves has been studied by electron microscopy and image analysis of negatively stained particles. Over 1500 molecular projections were selected and classified by multivariate statistical analysis. It was shown that the molecule consists of 14 subunits arranged in two layers with 72 point group symmetry. Side view projections of the molecule show a four-striation appearance, which subdivides both layers of seven subunits into two halves; this may be explained by a two-domain structure of the subunits. The presence in protein preparations of projections corresponding to one layer of subunits or half-molecules is consistent with the molecular structure suggested. Electron microscopic evidence for a specific association of GroEL-like protein and octameric glutamine synthetase, which was co-purified with this protein, was obtained.

The Vesicle Inducing Protein in Plastids 1 (Vipp1) was suggested to be involved in thylakoid membrane formation in both chloroplasts and cyanobacteria. The protein shows sequence homology to the Phage Shock Protein A (PspA) from bacteria, and both proteins have similar secondary structures. 2D-structures of PspA and of Vipp1 have been determined by electron microscopy in the recent years. Both PspA and Vipp1 form large homooligomeric rings with high molecular masses but their ring dimensions differ significantly. Furthermore, Vipp1 forms rings with different rotational symmetries whereas PspA appears to form rings with singular rotational symmetry. In this article addendum we compare the structures of PspA and Vipp1. Furthermore, we suggest a spatial structural model of the observed Vipp1 rings.

The vesicle-inducing protein in plastids 1 (Vipp1) was found to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. In contrast to chloroplasts, it has been suggested that in cyanobacteria the protein is only tightly associated with the cytoplasmic membrane. In the present study we analyze and describe the subcellular localization and the oligomeric organization of Vipp1 from the cyanobacterium Synechocystis PCC 6803. Vipp1 forms stable dimers and higher-ordered oligomers in the cytoplasm as well as at both the cytoplasmic and thylakoid membrane. Vipp1 oligomers are organized in ring structures with a variable diameter of 25–33 nm and corresponding calculated molecular masses of ∼1.6–2.2 MDa. Six different types of rings were found with an unusual 12–17-fold symmetrical conformation. The simultaneous existence of multiple types of rings is very unusual and suggests a special function of Vipp1. Involvement of diverse ring structures in vesicle formation is suggested.

The structure of three secondary transporter proteins, GltT of Bacillus stearothermophilus, CitS of Klebsiella pneumoniae, and GltS of Escherichia coli, was studied. The proteins were purified to homogeneity ill detergent solution by Ni(2+)-NTA affinity chromatography, and the complexes were determined by BN-PAGE to be trimeric, dimeric, and dimeric for GItT, CitS, and GItS, respectively. The subunit stoichiometry correlated with the binding affinity of the Ni(2+)-NTA resin for the protein complexes. Projection maps of negatively stained transporter particles were obtained by single-particle electron microscopy. Processing of the GltT particles revealed a projection map possessing 3-fold rotational symmetry, in good agreement with the trimer observed in the crystal structure of a homologous protein, Glt(Ph) of Pyrococcus horikoshii. The CitS protein showed up in two main views: as a kidney-shaped particle and a biscuit-shaped particle, both with a long axis of 160 angstrom. The latter has a width of 84 angstrom, the former of 92 angstrom. Symmetry considerations identify the biscuit shape as a top view, and the kidney shape as a side view from within the membrane. Combining the two images shows that the CitS dimer is a protein with a strong curvature at one side of the membrane and, at the opposite side, an indentation in the middle at the Subunit interface. The GltS protein was shaped like CitS with dimensions of 145 angstrom x 84 angstrom. The shapes and dimensions of the CitS and GItS particles are consistent with a similar structure of these two unrelated proteins.

The protein complexes of the respiratory chain interact by forming large protein particles called respiratory supercomplexes or "respirasomes". Biochemical characterization of these particles proved to be difficult because of their instability. Here we describe a strategy to isolate and characterize the cytochrome c reductase/cytochrome c oxidase supercomplex of yeast, also termed the III + IV supercomplex, which is based on lactate cultivation of yeast, gentle isolation of mitochondria, membrane solubilization by digitonin, sucrose gradient ultracentrifugation, and native gel electrophoresis. The procedure yields pure forms of two varieties of the III + IV supercomplex composed of dimeric complex III and one or two copies of monomeric complex IV. Supercomplex preparations can be used for physiological or structural investigations.

The protein complexes of the respiratory chain interact by forming large protein particles called respiratory supercomplexes or "respirasomes". Biochemical characterization of these particles proved to be difficult because of their instability. Here we describe a strategy to isolate and characterize the cytochrome c reductase/cytochrome c oxidase supercomplex of yeast, also termed the III + IV supercomplex, which is based on lactate cultivation of yeast, gentle isolation of mitochondria, membrane solubilization by digitonin, sucrose gradient ultracentrifugation, and native gel electrophoresis. The procedure yields pure forms of two varieties of the III + IV supercomplex composed of dimeric complex III and one or two copies of monomeric complex IV. Supercomplex preparations can be used for physiological or structural investigations.

This review describes aspects of negative staining of isolated integral membrane proteins. Detergents play a central role in the isolation of membrane proteins and also in their solubility in aqueous solutions. Specimens of mixed micelles of membrane proteins and nonionic detergents can be easily prepared as long as the detergent concentration remains above the critical micellar concentration. Membrane proteins involved in the process of photosynthesis have been taken as examples to illustrate their interaction with different detergents. Upon negative staining, mixed micelles of membrane proteins and detergents show characteristic top and side view projections. On their sides, mixed micelles can easily aggregate into strings.

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

Amphiphilic molecules possess parts which are distinctively hydrophobic and other parts which are distinctively hydrophilic. This ambivalence lies at the basis of the intriguing properties of these molecules, selected by nature as the building-blocks of architectures essential for life. Amphiphile self-assembly can lead to a variety of structures, differing in shape and size, depending on the molecular structure of the amphiphile and on the solution conditions such as temperature, ionic strength and pH. A relatively new and particularly interesting class of amphiphilic molecules, the gemini surfactants (GS), is obtained by connecting two single-tailed surfactants via a spacer at the level of the headgroups. The sugar-based gemini surfactant consists of several unique structural elements by which self-assembly properties can be made to fit the desired properties. The tertiary amino moieties in the headgroup can be protonated depending on the solution pH. The reduced sugars can be varied in length and stereochemistry. The spacer is either hydrophobic or hydrophilic and can be varied in length and the hydrophobic alkyl chain can be varied in length and degree of saturation. Most gemini surfactants form vesicles around neutral pH and exhibit a phase change towards micelles upon decreasing the pH, figure 1. When the nitrogens are protonated the cross-sectional headgroup area increases which gives the molecule a cone-shape appearance. The pH at which the phase changes from lamellar to micellar is dependent on the spacer length [1]. The shorter the spacer the more difficult it is to protonate both nitrogens which gives a lower pH of transition. At high pH the vesicles become neutral which leads to a decreased colloidal stability and the vesicles aggregated.

Peroxisomal matrix proteins are synthesized on free polyribosomes and directed to the organelle by specific peroxisomal targeting signals (PTSs). The PEX5 gene encodes the PTS1 receptor, Pex5p, which interacts with the PTS1 signal via a series of tetratricopeptide repeats (TPRs) within its C terminus. A crystal structure has been determined of a 41 kDa fragment of human Pex5p that includes six TPR motifs in complex with a small peptide containing a PTS1 sequence [1,2] or the sterol carrier protein [3]. This structure reveals the molecular basis for PTS1 recognition which is mostly formed by two clusters of three TPRs almost completely surrounding the PTS1-peptide. However, whether or not Pex5p functions as an oligomer, is still a matter of debate. Gel filtration chromatography and electron microscopy studies indicated that human Pex5p (HsPex5p) is a homotetramer [4]. Fluorescence spectroscopy studies on Pex5p of the yeast Hansenula polymorpha (HpPex5p) indicated that HpPex5p also forms oligomers [5]. In this study, the projection structures of HpPex5p and HpPex5p-HpPex20p complexes were investigated by single particle electron microscopy. The analysis shows that HpPex5p is a tetramer and that HpPex20p is able to induce a major conformational change leading to a rather open space in the centre of the HpPex5p tetramer. In a successive set of experiments we show that HpPex5p-HpPex20p complexes are able to bind folded copies of tetrameric catalase at the periphery. Since catalase is one of the major peroxisomal proteins this indicates that such HpPex5p-HpPex20p-catalase complexes are functional as receptor complex.

Self-assembly of small molecular components holds great promises as a bottom-up approach for nano-objects, but functionality of the resulting nanostructures can by far not compete with the sophisticated systems provided by nature. Surfactants, for instance, can lead to a great diversity of aggregates and mesophases (micelles, vesicles, cubic phases...), but with a level of complexity and functionality that still remains limited. Just like in nature, to increase the level of complexity in self-assembling systems, a straightforward approach consists in making use of multiple components that can display orthogonal self-assembly -i.e. the independent formation of two supramolecular structures each with their own characteristic within a single system. More precisely, we have associated surfactants with low-molecular weight hydrogelators: these molecules, based on cyclohexyl-tris-amino acid, can also self-assemble in one direction through the establishment of H-bonds, leading to the formation of a fiber network and consequently macroscopic gels. Work on mixing behavior of surfactants and various gelators have shown the independent formation of a fibrillar network with encapsulated spherical micelles, Figure 1. In order to produce even more complex nanostructures, this approach has been extended to worm-like micelles that can lead to viscoelastic gels, due to their entanglement. Interestingly, the formation of interpenetrating networks, with original and tuneable rheological properties, has been evidenced by cryo-TEM [1]. Screening of various gelators with vesicle-forming surfactants also revealed that most combinations display orthogonal self assembly, Figure 1.

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c(2) proteins were found. The most common complexes have Chl a/c(2) complexes at both sides of the PSI core monomer and have dimensions of about 17 x 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c(2) light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c(2) proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c(2) proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants. (C) 2008 Elsevier B.V. All rights reserved.

The structure of three types of Photosystem I (PS I) complex isolated from spinach chloroplasts was studied by electron microscopy and computer image analysis. Molecular projections (top views and side views) of a native PS I complex (PSI-200), an antenna-depleted PS I complex (PSI-100) and the PS I reaction center complex (CPI) were analyzed. The overall structure of the native PS I complex was found to be a disk with dimensions (corrected for attached detergent) of 16 × 12 nm in the plane of the membrane and a height of 6.8 nm. The PSI-100 and CPI complexes gave much smaller projections but these were similar in shape and size to those of the previously analyzed cyanobacterial PS I complex. The arrangement of the subunits within the native PS I complex is discussed. It is concluded that a shell of about eight light-harvesting complex (LHCI) subunits attached to the PSI-100 complex fits the dimensions and shape of the PSI-200 complex.

Photosystem I preparations from the cyanobacterium Synechococcus sp. were treated with high concentrations of Tris and octyl glucoside at alkaline pH and elevated temperature. A sucrose density gradient yielded three pigment-protein complexes; these were further purified on a HPLC anion-exchange column. In contrast to SDS-PAGE under non-denaturing conditions, this method produces homogeneous, highly active PS I particles in large quantities using mild solubilization conditions. Gel-filtration HPLC, SDS-PAGE, antenna Chl Chl a 1 ratio and electron microscopy images suggest that the three complexes represent monomeric, dimeric and trimeric forms of a minimal reaction center I unit. The size of this monomeric complex is 15.3 × 10.6 × 6.4 nm (length × width × height) as determined by electron microscopy. The apparent molecular mass - including 65 antenna chlorophylls per Chl a1, but excluding the detergent shell - is estimated by three different methods to be (235 ± 25) kDa.

This review summarizes the present literature on two-dimensional crystallization of membrane proteins, with emphasis on the technical aspects. It includes all the intrinsic membrane proteins that have been crystallized after solubilization. Four general ways of making crystals are described in detail. Furthermore, suggestions for improving crystallization conditions are presented.

Intact chlorosomes of Chlorobium tepidum were embedded in amorphous ice layers and examined by cryoelectron microscopy to study the long-range organization of bacteriochlorophyll ( BChl) layers. End-on views reveal that chlorosomes are composed of several multi-layer tubules of variable diameter (20-30 nm) with some locally undulating non-tubular lamellae in between. The multi-layered tubular structures are more regular and larger in a C. tepidum mutant that only synthesizes [8-ethyl, 12-methyl]-BChl d. Our data show that wild-type C. tepidum chlorosomes do not have a highly regular, long-range BChl c layer organization and that they contain several multilayered tubules rather than single-layer tubules or exclusively undulating lamellae as previously proposed. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Green plant photosystem I (PS I) not only binds a chlorophyll a/b-binding, membrane-intrinsic antenna complex (LHCI) that is associated with the PS I core complex under almost all physiological conditions, but it can also transiently bind the major chlorophyll a/b-binding light-harvesting complex (LHCII), when the light conditions favor excitation of photosystem II (PS II) and the photosynthetic apparatus is in the so-called state 2. Recently, a low-resolution structure was obtained of a PS I–LHCII supercomplex from Arabidopsis thaliana. We describe here some of the structural features of this transient complex, and discuss the role of small PS I subunits that are involved in the binding of LHCII. We also discuss structural features of the PS I complex of the green algae Chlamydomonas reinhardtii, which has a larger LHCI antenna and shows a more pronounced difference between state 1 and state 2.

Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the 'red' chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI. (c) 2007 Elsevier B.V. All rights reserved.

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.

Mitochondrial respiratory chain complexes are arranged in supercomplexes within the inner membrane. Interaction of cytochrome c reductase ( complex III) and cytochrome c oxidase ( complex IV) was investigated in Saccharomyces cerevisiae. Projection maps at 15 angstrom resolution of supercomplexes III2 + IV1 and III2 + IV2 were obtained by electron microscopy. Based on a comparison of our maps with atomic x-ray structures for complexes III and IV we present a pseudo-atomic model of their precise interaction. Two complex IV monomers are specifically attached to dimeric complex III with their convex sides. The opposite sides, which represent the complex IV dimer interface in the x-ray structure, are open for complex IV-complex IV interactions. This could lead to oligomerization of III2 + IV2 supercomplexes, but this was not detected. Instead, binding of cytochrome c to the supercomplexes was revealed. It was calculated that cytochrome c has to move less than 40 angstrom at the surface of the supercomplex for electron transport between complex III2 and complex IV. Hence, the prime function of the supercomplex III2 + IV2 is proposed to be a scaffold for effective electron transport between complexes III and IV.

The proton ATP synthase consists of a membrane integrated part, Fo, and a hydrophilic part, F1. F1 is composed of five different subunits: α, β, γ, δ and ε. This chapter focuses on the chloroplast F1 (CF1) structure and discusses the overall shape and dimensions of CF1, shape and size of the various subunits, subunit interactions and conformational changes in the subunit positions related to catalysis. Structural data originate mainly from X-ray diffraction and electron microscopy. Recently, the structure of F1 from beef heart mitochondria has been determined at 2.8 Å and this structure, although not fully identical to CF1, can be taken as a blueprint for models of the CF1 structure.

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-alpha,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes.

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO2 conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO2 growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.

Functional flexibility of light harvesting in cyanobacteria Nataliya Yeremenko 1, Roman Kouril 2, Janne A. Ihalainen 3, Sandrine D¿Haene 3, Martin Hagemann 4, Egbert J. Boekema 2, Hans C.P. Matthijs 1 and Jan P. Dekker 31 Universiteit van Amsterdam, Amsterdam, The Netherlands; 2 University of Groningen, Groningen, The Netherlands; 3 Vrije Universiteit, Amsterdam, The Netherlands; 4 Universität Rostock, Rostock, Germany Regulation of light harvesting is a matter of high importance in photosynthetic organisms. High plants and algae involve mechanism of state transitions which is regulated by the redox state of the plastoquinone pool and leads to the reversible redistribution of excitation energy between the two photosystems through a reorganization of the antennae and to an overall increase in photosynthetic quantum yield by decreasing the delivery of excess excitation energy to PSII while increasing delivery of excitation energy to PSI (or vice-versa) [1]. Cyanobacteria balance energy input and consumption differently. Under iron-deficient conditions, the content of PSI decreases substantially more than that of PSII [2]. Cyanobacteria respond to this condition by accumulation of IsiA protein which may occurs in two different types of complexes (associated and not associated with PSI) and correspondingly serve different functions [3]. The PSI-IsiA supercomplexes show increasingly larger amounts of bound IsiA proteins with increasing iron starvation. The largest particles consist of double IsiA ring around monomeric PSI and contain about 560 chlorophylls, which together with 96 chlorophylls of the PSI core complex would give a total number of 656 chlorophylls. PSI particles with such large antenna sizes have not been observed before. Fluorescence excitation spectra indicate an efficient light-harvesting function for all PSI-bound chlorophylls. With continious iron starvation the further decline of PSI makes PSII increasingly vulnerable to photooxidation and the surplus synthesis of IsiA not associated with PSI shields PSII from excess light.

Chloroplasts are central to the provision of energy for green plants. Their photosynthetic membrane consists of two major complexes converting sunlight: photosystem I (PSI) and photosystem II (PSII). The energy flow toward both photosystems is regulated by light-harvesting complex II (LHCII), which after phosphorylation can move from PSII to PSI in the so-called state 1 to state 2 transition and can move back to PSII after dephosphorylation. To investigate the changes of PSI and PSII during state transitions, we studied the structures and frequencies of all major membrane complexes from Arabidopsis thaliana chloroplasts at conditions favoring either state 1 or state 2. We solubilized thylakoid membranes with digitonin and analyzed the complete set of complexes immediately after solubilization by electron microscopy and image analysis. Classification indicated the presence of a PSI-LHCII supercomplex consisting of one PSI-LHCI complex and one LHCII trimer, which was more abundant in state 2 conditions. The presence of LHCII was confirmed by excitation spectra of the PSI emission of membranes in state 1 or state 2. The PSI-LHCII complex could be averaged with a resolution of 16 Å, showing that LHCII has a specific binding site at the PSI-A, -H, -L, and -K subunits. © 2005 American Chemical Society.

Large data sets of molecular projections of the membrane proteins Photosystem I and Photosystem II from cyanobacteria were analyzed by single particle electron microscopy (EM). Analysis resulted in the averaging of 2D projections from the purified complexes but also in the simultaneous detection and averaging of 2D projections from large contaminating complexes, which were present in frequencies as low as 0.1%. Among them T-shaped and L-shaped contaminants were found. The L-shaped particles could be assigned to Complex I just from the shape, although no Complex I from a cyanobacterium has been structurally characterized. A systematic comparison by single particle EM and mass spectrometry of two differently purified Photosystem II complexes resulted in the assignment of PsbZ, a small peripheral subunit of 6.8 kDa, within the structure. Together these data suggest that screening for membrane protein structures by single particle EM and mass spectrometry may be a new approach to find novel structures of such proteins. We propose here a scheme for searching for novel membrane protein structures in specific types of membranes. In this approach single particle EM and mass spectrometry, after pre-fractionation using one- or multidimensional protein separation techniques, are applied to characterize all its larger components.

Co-reconstitution of subunits E and G of the yeast V-ATPase and the alpha and beta subunits of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) resulted in an alpha(3)beta(3)EG hybrid complex showing 53% of the ATPase activity of TF(1). The alpha(3)beta(3)EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2-2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the alpha(3)beta(3) subcomplex from TF(1) (Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) Structure 5, 825-836) demonstrates that a seventh mass is located inside the shaft of the alpha(3)beta(3) barrel and extends out from the hexamer. Furthermore, difference mapping of the alpha(3)beta(3)EG oligomer with projections of the A(3)B(3)E and A(3)B(3)EC subcomplexes of the V(1) from Caloramator fervidus (Chaban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO Rep. 3, 982-987) shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active alpha(3)beta(3)EG hybrid complex indicates that the coupling subunit gamma inside the alpha(3)beta(3) oligomer of F(1) can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and gamma subunits are structurally similar, despite the fact that their genes do not show significant homology.

The overall structure of V-ATPase complexes resembles that of F-type ATPases, but the stalk region is different and more complex. Database searches followed by sequence analysis of the five water-soluble stalk region subunits C-G revealed that (i) to date V-ATPases are found in 16 bacterial species, (ii) bacterial V-ATPases are closer to archaeal A-ATPases than to eukaryotic V-ATPases, and (iii) different groups of bacterial V-ATPases exist. Inconsistencies in the nomenclature of types and subunits are addressed. Attempts to assign subunit positions in V-ATPases based on biochemical experiments, chemical cross-linking, and electron microscopy are discussed. A structural model for prokaryotic and eukaryotic V-ATPases is proposed. The prokaryotic V-ATPase is considered to have a central stalk between headpiece and membrane flanked by two peripheral stalks. The eukaryotic V-ATPases have one additional peripheral stalk.

The ntpK gene of the archaeon Methanopyrus kandleri encodes the equivalent of the c subunit of ATP synthase. The gene product contains 1021 residues and consists of 13 homologous domains, each one corresponding to a single helical hairpin. The amino acid sequence of the domains is highly conserved, ranging between 50 and 80% sequence identity. Each of the 13 domains contains a conserved Gln and Glu residue in the N- and C-terminal helix, respectively, both of which are believed to be involved in cation binding. The protein is likely to form the monomeric rotor of the ATP synthase that consists of 13 hairpin domains. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

The Na+-pumping V-ATPase complex of the thermophilic bacterium Caloramator fervidus was purified and dissociated under controlled conditions. The structure of purified V-1-ATPase subcomplexes differing in subunit composition was analyzed by electron microscopy and single particle analysis of 50 000 projections. Difference mapping of subcomplex projections revealed the presence and position of two subunits in the central stalk. A density with an elongated shape similar to the gamma subunit of F-ATPases is partly located within V-1 and corresponds, most likely, to subunit E. Subunit E is connected to the membrane-bound part V-0 via subunit C, a spherical density that is connected to the center of V-0. The presence of subunit C makes the central stalk substantially longer in comparison to the F-ATPases, in which the subunit connects directly to F-0.

Cyanobacteria are abundant throughout most of the world's water bodies and contribute significantly to global primary productivity through oxygenic photosynthesis. This reaction is catalysed by two membrane-bound protein complexes, photosystem I (PSI) and photosystem II (PSII), which both contain chlorophyll-binding subunits functioning as an internal antenna1. In addition, phycobilisomes act as peripheral antenna systems, but no additional light-harvesting systems have been found under normal growth conditions. Iron deficiency, which is often the limiting factor for cyanobacterial growth in aquatic ecosystems2, leads to the induction of additional proteins such as IsiA (ref. 3). Although IsiA has been implicated in chlorophyll storage, energy absorption and protection against excessive light, its precise molecular function and association to other proteins is unknown. Here we report the purification of a specific PSI–IsiA supercomplex, which is abundant under conditions of iron limitation. Electron microscopy shows that this supercomplex consists of trimeric PSI surrounded by a closed ring of 18 IsiA proteins binding around 180 chlorophyll molecules. We provide a structural characterization of an additional chlorophyll-containing, membrane-integral antenna in a cyanobacterial photosystem.