Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å

Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 ?. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 ?. From our electron density map, we located all of the metal atoms of the Mn(4)CaO(5) cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn(4)CaO(5) cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.     

Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II

Oxygenic photosynthesis in plants, algae and cyanobacteria is initiated at photosystem II, a homodimeric multisubunit protein-cofactor complex embedded in the thylakoid membrane. Photosystem II captures sunlight and powers the unique photo-induced oxidation of water to atmospheric oxygen. Crystallographic investigations of cyanobacterial photosystem II have provided several medium-resolution structures (3.8 to 3.2 A) that explain the general arrangement of the protein matrix and cofactors, but do not give a full picture of the complex. Here we describe the most complete cyanobacterial photosystem II structure obtained so far, showing locations of and interactions between 20 protein subunits and 77 cofactors per monomer. Assignment of 11 beta-carotenes yields insights into electron and energy transfer and photo-protection mechanisms in the reaction centre and antenna subunits. The high number of 14 integrally bound lipids reflects the structural and functional importance of these molecules for flexibility within and assembly of photosystem II. A lipophilic pathway is proposed for the diffusion of secondary plastoquinone that transfers redox equivalents from photosystem II to the photosynthetic chain. The structure provides information about the Mn4Ca cluster, where oxidation of water takes place. Our study uncovers near-atomic details necessary to understand the processes that convert light to chemical energy.     

Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution.

Life on Earth depends on photosynthesis, the conversion of light energy from the Sun to chemical energy. In plants, green algae and cyanobacteria, this process is driven by the cooperation of two large protein-cofactor complexes, photosystems I and II, which are located in the thylakoid photosynthetic membranes. The crystal structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus described here provides a picture at atomic detail of 12 protein subunits and 127 cofactors comprising 96 chlorophylls, 2 phylloquinones, 3 Fe4S4 clusters, 22 carotenoids, 4 lipids, a putative Ca2+ ion and 201 water molecules. The structural information on the proteins and cofactors and their interactions provides a basis for understanding how the high efficiency of photosystem I in light capturing and electron transfer is achieved.     

Crystal structure of plant photosystem I

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on Earth. The conversion of sunlight into chemical energy is driven by two multisubunit membrane protein complexes named photosystem I and II. We determined the crystal structure of the complete photosystem I (PSI) from a higher plant (Pisum sativum var. alaska) to 4.4 A resolution. Its intricate structure shows 12 core subunits, 4 different light-harvesting membrane proteins (LHCI) assembled in a half-moon shape on one side of the core, 45 transmembrane helices, 167 chlorophylls, 3 Fe-S clusters and 2 phylloquinones. About 20 chlorophylls are positioned in strategic locations in the cleft between LHCI and the core. This structure provides a framework for exploration not only of energy and electron transfer but also of the evolutionary forces that shaped the photosynthetic apparatus of terrestrial plants after the divergence of chloroplasts from marine cyanobacteria one billion years ago.     

Structure of fumarate reductase from Wolinella succinogenes at 2.2 A resolution

Fumarate reductase couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalysed by the related complex II of the respiratory chain (succinate dehydrogenase). Here we describe the crystal structure at 2.2 A resolution of the three protein subunits containing fumarate reductase from the anaerobic bacterium Wolinella succinogenes. Subunit A contains the site of fumarate reduction and a covalently bound flavin adenine dinucleotide prosthetic group. Subunit B contains three iron-sulphur centres. The menaquinol-oxidizing subunit C consists of five membrane-spanning, primarily helical segments and binds two haem b molecules. On the basis of the structure, we propose a pathway of electron transfer from the dihaem cytochrome b to the site of fumarate reduction and a mechanism of fumarate reduction. The relative orientations of the soluble and membrane-embedded subunits of succinate:quinone oxidoreductases appear to be unique.     

Detection of an intermediate of photosynthetic water oxidation

The oxygen that we breathe is produced by photosystem II of cyanobacteria and plants. The catalytic centre, a Mn4Ca cluster, accumulates four oxidizing equivalents before oxygen is formed, seemingly in a single reaction step 2H2O<==>O2 + 4H+ + 4e-. The energy and cycling of this reaction derives solely from light. No intermediate oxidation product of water has been detected so far. Here, we shifted the equilibrium of the terminal reaction backward by increasing the oxygen pressure and monitoring (by absorption transients in the near-ultraviolet spectrum) the electron transfer from bound water into the catalytic centre. A tenfold increase of ambient oxygen pressure (2.3 bar) half-suppressed the full progression to oxygen. The remaining electron transfer at saturating pressure (30 bar) was compatible with the formation of a stabilized intermediate. The abstraction of four electrons from water was probably split into at least two electron transfers: mildly endergonic from the centre's highest oxidation state to an intermediate, and exergonic from the intermediate to oxygen. There is little leeway for photosynthetic organisms to push the atmospheric oxygen concentration much above the present level.     

Novel thioether bond revealed by a 1.7 A crystal structure of galactose oxidase

Galactose oxidase is an extracellular enzyme secreted by the fungus Dactylium dendroides. It is monomeric, with a relative molecular mass of 68,000, catalyses the stereospecific oxidation of a broad range of primary alcohol substrates and possesses a unique mononuclear copper site essential for catalysing a two-electron transfer reaction during the oxidation of primary alcohols to corresponding aldehydes. Recent evidence arguing against a Cu(III)-Cu(I) couple implies the existence of a second redox-active site proposed to involve pyrroloquinoline quinone or a tyrosine radical. We now report the crystal structure of galactose oxidase at 1.7 A resolution. This reveals a unique structural feature at the copper site with a novel thioether bond linking Cys 228 and Tyr 272 in a stacking interaction with Trp 290. We propose that these molecular components stabilize the protein free-radical species essential for catalysis and thus provide a 'built-in' secondary cofactor. This feature may represent a new mechanism for mediating electron transfer in metalloenzymes in the absence of exogenous cofactors.     

Methanol as a probe to investigate the relationship between the secondary electron donor TyrZ and the substrate water molecules in active photosystem Ⅱ

The secondary electron donor, TyrZ, is implicated in tuning the primary charge separation and the water oxidation in active pho-tosystem II (PSII). Two types of mechanisms have been proposed to explain the function of TyrZ. One is that TyrZ tunes the water oxidation through the direct interaction with substrate water molecules; the other is that TyrZ is located in a hydrophobic envi-ronment without interacting with H2O, and controls the water oxidation by tuning the strength of the hydrogen bond between Tyr...     

Methanol as a probe to investigate the relationship between the secondary electron donor Tyr<Subscript>Z</Subscript> and the substrate water molecules in active photosystem II

The secondary electron donor, TyrZ, is implicated in tuning the primary charge separation and the water oxidation in active pho-tosystem II (PSII). Two types of mechanisms have been proposed to explain the function of TyrZ. One is that TyrZ tunes the water oxidation through the direct interaction with substrate water molecules; the other is that TyrZ is located in a hydrophobic envi-ronment without interacting with H2O, and controls the water oxidation by tuning the strength of the hydrogen bond between TyrZ and His190. Here, methanol was used as a probe to study the possible relationship between TyrZ and H2O by monitoring the TyrZ oxidation and TyrZ· reduction at cryogenic temperatures with electron paramagnetic resonance spectroscopy. The oxidation of TyrZ and reduction of TyrZ· in both S2 and S0 states at 10 K were accelerated by addition of a small amount of methanol (6%). Theoretical studies indicate that Tyr oxidation becomes more difficult if it interacts directly with the methanol molecule; while the decrease of the polarity of its environment accelerates the oxidation of Tyr. Accordingly, CH3OH does not directly interact with TyrZ in active PSII, and the accelerative effect of methanol is caused by the strength increase of the hydrogen bond between TyrZ and His190, resulting from the decrease of polarity of their environment after the displacement of H2O by CH3OH inside PSII. Considering the similarity between methanol and water, the results in this study support the model in which TyrZ does not interact with H2O in active PSII.     

Structure of the 30S ribosomal subunit

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.     

Sliding-layer conformational change limited by the quaternary structure of plant RuBisCO

RuBisCO, D-ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39), converts carbon dioxide to sugar in the first step of photosynthesis. In plants and some bacteria, this enzyme has an L8S8 structure, where L is the large catalytic subunit and S is the small subunit of unknown function. The molecule resembles a keg 105 A along the 4-fold axis and 132 A in diameter at the widest point of the keg. Here we describe the quaternary structure of RuBisCO from N. tabacum, the first L8S8 type known from an X-ray crystallographic study at near-atomic resolution (3 A). The structure shows that all eight L subunits are elongated along the 4-fold axis so that the molecule cannot be simply described as layers of subunits, as it had been from studies by electron microscopy. The structure, with its elongated and interdigitated L subunits, is evidence against a large, sliding-layer conformational change in plant RuBisCO, as proposed recently in Nature for the same enzyme from Alcaligenes eutrophus.     

等规聚苯乙烯单链单晶的结构和耐辐照性能

利用电子衍射（ED）和高分辨电子显微（HREM）技术，研究等规聚苯乙烯（i－PS）单链单晶的结构．纳米级的单链单晶具有很强的耐电子辐照性能．按照i－PS的晶胞能对ED图中的衍射环和HREM像中的晶格条纹进行晶面指标化，但发现低指数衍射缺失．由于单链单晶的尺寸很小，电子辐照所产生的次级电子可以逸出晶体，使辐照损伤大大减小．另外，单链单晶存在着较少的低指数晶面，未能产生足够的衍射强度，使低指数晶面的衍射缺失．单链单晶对电子辐照稳定，在室温下，可得到高分辨晶格条纹象，这为研究高分子晶体的结构开辟了新的实验方法．     

The structure of the protein disk of tobacco mosaic virus to 5A resolution.

An electron density map of the TMV disk at 5A resolution has been obtained using isomorphous replacement and non-crystallographic symmetry. The polypeptide chain can be traced with little ambiguity. The axial contacts between protein subunits are unlike those in the virus, the disk being a more open structure apparently designed for rapid interaction with the RNA.     

The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre

Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H(2) into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O(2) are considered to be central to H(2)-based technologies, such as enzymatic fuel cells and for light-driven H(2) production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O(2) has remained unclear. Here we present the crystal structure of an O(2)-tolerant [NiFe]-hydrogenase from the aerobic H(2) oxidizer Ralstonia eutropha H16 at 1.5?? resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H(2)-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H(2) oxidation and as an electron-delivering device upon O(2) attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H(2)-converting catalysts that are capable of cycling H(2) in air.     

Lipid-protein interactions in double-layered two-dimensional AQP0 crystals

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.     

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.     

Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes

The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time.     

X-ray structure of a prokaryotic pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) are key players in the early events of electrical signal transduction at chemical synapses. The family codes for a structurally conserved scaffold of channel proteins that open in response to the binding of neurotransmitter molecules. All proteins share a pentameric organization of identical or related subunits that consist of an extracellular ligand-binding domain followed by a transmembrane channel domain. The nicotinic acetylcholine receptor (nAChR) is the most thoroughly studied member of the pLGIC family (for recent reviews see refs 1-3). Two sources of structural information provided an architectural framework for the family. The structure of the soluble acetylcholine-binding protein (AChBP) defined the organization of the extracellular domain and revealed the chemical basis of ligand interaction. Electron microscopy studies of the nAChR from Torpedo electric ray have yielded a picture of the full-length protein and have recently led to the interpretation of an electron density map at 4.0 A resolution. Despite the wealth of experimental information, high-resolution structures of any family member have so far not been available. Until recently, the pLGICs were believed to be only expressed in multicellular eukaryotic organisms. The abundance of prokaryotic genome sequences, however, allowed the identification of several homologous proteins in bacterial sources. Here we present the X-ray structure of a prokaryotic pLGIC from the bacterium Erwinia chrysanthemi (ELIC) at 3.3 A resolution. Our study reveals the first structure of a pLGIC at high resolution and provides an important model system for the investigation of the general mechanisms of ion permeation and gating within the family.     

Determination of surface topography of biological specimens at high resolution by scanning tunnelling microscopy

Although techniques are available for the determination of the three-dimensional structure of biological specimens, for example scanning electron microscopy, they all have some serious drawback, such as low resolution, the requirement for crystals or for the sample to be analysed in a high vacuum. In an attempt to develop a technique for high-resolution three-dimensional structure analysis of non-crystalline biological material, we have tested the applicability of scanning tunnelling microscopy (STM), a method that has been used successfully in the analysis of metal and semiconductor surface structures. We report here that scanning tunnelling electron microscopy can be used to determine the surface topography of biological specimens at atmospheric pressure and room temperature, giving a vertical resolution of the order of 1 A. Our results show that quantum mechanical tunnelling of electrons through biological material is possible provided that the specimen is deposited on a conducting surface.