Plants lacking the main light-harvesting complex retain photosystem II macro-organization.

Photosystem II (PSII) is a key component of photosynthesis, the process of converting sunlight into the chemical energy of life. In plant cells, it forms a unique oligomeric macrostructure in membranes of the chloroplasts. Several light-harvesting antenna complexes are organized precisely in the PSII macrostructure-the major trimeric complexes (LHCII) that bind 70% of PSII chlorophyll and three minor monomeric complexes-which together form PSII supercomplexes. The antenna complexes are essential for collecting sunlight and regulating photosynthesis, but the relationship between these functions and their molecular architecture is unresolved. Here we report that antisense Arabidopsis plants lacking the proteins that form LHCII trimers have PSII supercomplexes with almost identical abundance and structure to those found in wild-type plants. The place of LHCII is taken by a normally minor and monomeric complex, CP26, which is synthesized in large amounts and organized into trimers. Trimerization is clearly not a specific attribute of LHCII. Our results highlight the importance of the PSII macrostructure: in the absence of one of its main components, another protein is recruited to allow it to assemble and function.

Supermolecular organization of photosystem II and its associated light-harvesting antenna in Arabidopsis thaliana.

The organization of Arabidopsis thaliana photosystem II (PSII) and its associated light-harvesting antenna (LHCII) was studied in isolated PSII-LHCII supercomplexes and native membrane-bound crystals by transmission electron microscopy and image analysis. Over 4000 single-particle projections of PSII-LHCII supercomplexes were analyzed. In comparison to spinach supercomplexes [Boekema, E.J., van Roon, H., van Breemen, J.F.L. & Dekker, J.P. (1999) Eur. J. Biochem. 266, 444-452] some striking differences were revealed: a much larger number of supercomplexes from Arabidopsis contain copies of M-type LHCII trimers. M-type trimers can also bind in the absence of the more common S-type trimers. No binding of l-type trimers could be detected. Analysis of native membrane-bound PSII crystals revealed a novel type of crystal with a unit cell of 25.6 x 21.4 nm (angle 77 degrees ), which is larger than any of the PSII lattices observed before. The data show that the unit cell is built up from C2S2M2 supercomplexes, rather than from C2S2M supercomplexes observed in native membrane crystals from spinach [Boekema, E.J., Van Breemen, J.F.L., Van Roon, H. & Dekker, J.P. (2000) J. Mol. Biol. 301, 1123-1133]. It is concluded from both the single particle analysis and the crystal analysis that the M-type trimers bind more strongly to PSII core complexes in Arabidopsis than in spinach.

Structure of the major membrane protein complex from urinary bladder epithelial cells by cryo-electron crystallography.

Numerous protein plaques cover the apical surface of mammalian urinary bladder epithelial cells. These plaques contain four integral membrane proteins, called uroplakins, which form a well-ordered array of hexameric complexes. The 3D structure of these naturally occurring 2D crystals was studied by cryo-electron-crystallographic methods using a slow-scan charged-coupled device (CCD) camera to record the electron micrographs. A 1.2 nm projection map calculated from untilted crystals shows that each hexamer comprises a ring of six inner and six outer domains at a radius of 5.7 nm and 9.2 nm respectively. The 3D structure shows that the mass is distributed strongly asymmetrically with respect to the membrane, with most of the mass protruding from the luminal face. Both domains in the asymmetric unit traverse the membrane and protrude from the membrane on the cytoplasmic side. On the luminal side, the two domains are bridged forming a stretched arc. The total thickness of the complex is about 13.2 nm. A model of the urothelial plaque reveals that contacts between the hexamers are much less extended than within the hexamers.

A giant chlorophyll-protein complex induced by iron deficiency in cyanobacteria.

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 antenna. 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 ecosystems, 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.

Structure of membrane-bound annexin A5 trimers: a hybrid cryo-EM - X-ray crystallography study.

Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to the common view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion.

The 5 A projection structure of the transmembrane domain of the mannitol transporter enzyme II.

The uptake of mannitol in Escherichia coli is controlled by the phosphoenolpyruvate dependent phosphotransferase system. Enzyme II mannitol (EIIMtl) is part of the phosphotransferase system and consists of three covalently bound domains. IICMtl, the integral membrane domain of EIIMtl, is responsible for mannitol transport across the cytoplasmic membrane. In order to understand this molecular process, two-dimensional crystals of IICMtl were grown by reconstitution into lipid bilayers and their structure was investigated by cryo-electron crystallography. The IICMtl crystals obey p22121 symmetry and have a unit cell of 125 Ax65 A, gamma=90 degrees. A projection structure was determined at 5 A resolution using both electron images and electron diffractograms. The unit cell contains two IICMtl dimers with a size of about 40 Ax90 A, which are oriented up and down in the crystal. Each monomer exhibits six domains of high density which most likely correspond to transmembrane alpha-helices and cytoplasmic loops.

A 7.4-A projection structure of outer membrane phospholipase A from Escherichia coli by electron crystallography.

Outer membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Escherichia coli. Two-dimensional crystals of OMPLA were grown by reconstitution of purified protein into lipid bilayers via detergent dialysis and were studied by electron crystallography. A 7.4-A projection map reveals OMPLA molecules exhibiting an oval-shaped domain of 30 x 20 A resembling the beta-barrel structure characteristic of porins, which is associated with a 25-A elongated domain of lower density.

The 1.5-nm projection structure of HeLa cell prosome-MCP (proteasome) provided by two-dimensional crystals.

We grew two-dimensional crystals of HeLa cell prosomes, also called multicatalytic proteinases (MCP) and proteasomes, for a structure determination by electron microscopy. The molecules were arranged in side views in these crystals. The crystals have p21 plane group symmetry with one particle per unit cell. This symmetry confirms previously published evidence indicating that eukaryotic prosome-MCPs are dimers of two identical halves. Structure factors from six crystals each comprising more than 1000 unit cells were combined to generate a 1.5-nm projection map. We discovered that while the general cylindrical shape of HeLa prosome-MCPs resembles the shape of the archaebacterial Thermoplasma acidophilum proteasomes, the internal structure differs significantly. We propose that because of different subunit composition, the architecture of HeLa prosome-MCPs differs from the basic architecture of related particles previously reported.

Crystallization properties and structure of Panulirus interruptus haemocyanin.

Electron-microscopic studies revealed that two types of subunits of Panulirus interruptus haemocyanin crystallize in different ways. Homohexamers of subunit a give close-packed two-dimensional crystals whereas homohexamers of subunit c form open two-dimensional arrays. We applied computer-image analysis to these arrays and studied the differences in crystallization properties by combining the electron-microscopic data with amino acid sequence information and the X-ray diffraction model of subunit a.

Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. I. Comparison of crystal structures in various negative stains.

We investigated the structure of two-dimensional crystals from bovine heart mitochondrial NADH: ubiquinone oxidoreductase. A detailed description of uranyl acetate-stained crystals demonstrated that they are composed of fragments in a spatial arrangement according to space group P4212 [J. Brink, S. Hovmöller, C.I. Ragan, M.W.J. Cleeter, E.J. Boekema and E.F.J. van Bruggen, European J. Biochem. 166 (1987) 287]. To gain more structural information on the crystal structure and to assess the effects of various negative stains on the structure preservation and appearance, we examined stained crystals by means of electron microscopy and image analysis. The space group P4212 appeared to be present for several stains tested, i.e. ammonium molybdate, uranyl acetate, uranyl nitrate and uranyl sulphate. Use of phosphotungstic acid and silicotungstate resulted in a reduction of symmetry to pseudo-P4212 or p4. Use of sodium tungstate led to a considerable loss of resolution to 3.8 nm at best, whereas otherwise 1.5 to 1.9 nm could be demonstrated. The lattice vectors were not affected by the stains; they were determined as a = b = 14.9 +/- 0.25 nm with gamma = 89.8 degrees +/- 0.6 degrees. Image analysis showed the presence of similar structures with the molybdate and uranyl compounds. Differences were observed in the case of the tungstate type of stains. Furthermore, the analysis revealed the complete absence of the four small pores of 2.0 nm diameter in the unit cell. This effect was observed irrespective of the type of stain and supporting film, and could be ascribed only to the glow-discharge treatment of the supporting film. The observed difference must be caused by changed interactions between the protein, stain and supporting film. Application of correspondence analysis and clustering algorithms to the various reconstructed images of the crystals showed that they could be separated into several clusters. Each of these clusters corresponded on the average to only one type of stain, whereas a further division according to the specific uranyl compounds was observed. This study therefore shows that under identical preparation conditions subtle differences between individual stains can be detected.

Two-dimensional crystallization experiments.

Our experience in the growth of two-dimensional crystals of different proteins is presented. Polyethylene glycol was used to produce two-dimensional arrays of haemocyanin from O. vulgaris and of cholera toxin. The arrays showed a hexagonal close-packed structure of only randomly oriented molecules. The increase in protein concentration probably occurred too quickly to allow complete crystallization. Different two-dimensional arrays of hexameric haemocyanin molecules (from P. interruptus) were obtained by microdialysis through the specimen supporting film. A comparison was made with X-ray data. Two-dimensional tetrameric arrays of molecules, possibly rhodopsin, were seen in samples of bovine retinal rod outer segments in the presence of ammonium sulphate. Two-dimensional crystals of complex I (from bovine mitochondria) were prepared by dialysis in the presence of ammonium sulphate. A three-dimensional reconstruction was made from two tilt-series by computer filtration using the direct SIRT procedure. Finally, the possibility of computer crystallization using correlation techniques in combination with correspondence analysis is discussed.

Two-dimensional crystallization of bovine rhodopsin.

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.

Structure of NADH:Q oxidoreductase from bovine heart mitochondria studied by electron microscopy.

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% (w/v) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 +/- 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.

Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.