Crystal structure of the ribosome at 5.5 A resolution

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.     

Structures of the bacterial ribosome in classical and hybrid states of tRNA binding

During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of ~3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.     

2.5岁及3.5岁麦洼犊牦牛屠宰试验

为研究经全哺乳饲养后,2.5岁及3.5岁麦洼公犊牦牛的生长发育及肉质特性。对2.5及3.5岁麦洼公犊牦牛进行屠宰试验研究。结果表明,2.5岁和3.5岁犊牦牛的宰前重、体高和体斜长存在极显著差异,管围具有显著差异,而胸围无显著差异。头、蹄、皮重均具有极显著差异,而生殖器睾丸重无显著差异。心脏、肝脏和肾脏重具有极显著差异,肺脏重具有显著差异,而脾脏无显著差异。2.5岁和3.5岁犊牦牛的胴体重、净骨重、净肉重、净肉率、胴体产肉率和骨肉比均具有极显著差异,眼肌面积具有显著差异,而屠宰率无显著差异。全哺乳方式对2.5岁及3.5岁犊牦牛,生长发育补充效果明显,均有较高的宰前重,且产肉性能良好。     

Three-dimensional structure of poliovirus at 2.9 A resolution

The three-dimensional structure of poliovirus has been determined at 2.9 A resolution by x-ray crystallographic methods. Each of the three major capsid proteins (VP1, VP2, and VP3) contains a "core" consisting of an eight-stranded antiparallel beta barrel with two flanking helices. The arrangement of beta strands and helices is structurally similar and topologically identical to the folding pattern of the capsid proteins of several icosahedral plant viruses. In each of the major capsid proteins, the "connecting loops" and NH2- and COOH-terminal extensions are structurally dissimilar. The packing of the subunit "cores" to form the virion shell is reminiscent of the packing in the T = 3 plant viruses, but is significantly different in detail. Differences in the orientations of the subunits cause dissimilar contacts at protein-protein interfaces, and are also responsible for two major surface features of the poliovirion: prominent peaks at the fivefold and threefold axes of the particle. The positions and interactions of the NH2- and COOH-terminal strands of the capsid proteins have important implications for virion assembly. Several of the "connecting loops" and COOH-terminal strands form prominent radial projections which are the antigenic sites of the virion.     

Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution

Halorhodopsin, an archaeal rhodopsin ubiquitous in Haloarchaea, uses light energy to pump chloride through biological membranes. Halorhodopsin crystals were grown in a cubic lipidic phase, which allowed the x-ray structure determination of this anion pump at 1.8 angstrom resolution. Halorhodopsin assembles to trimers around a central patch consisting of palmitic acid. Next to the protonated Schiff base between Lys(242) and the isomerizable retinal chromophore, a single chloride ion occupies the transport site. Energetic calculations on chloride binding reveal a combination of ion-ion and ion-dipole interactions for stabilizing the anion 18 angstroms below the membrane surface. Ion dragging across the protonated Schiff base explains why chloride and proton translocation modes are mechanistically equivalent in archaeal rhodopsins.     

Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.     

The atomic structure of Mengo virus at 3.0 A resolution

The structure of Mengo virus, a representative member of the cardio picornaviruses, is substantially different from the structures of rhino- and polioviruses. The structure of Mengo virus was solved with the use of human rhinovirus 14 as an 8 A resolution structural approximation. Phase information was then extended to 3 A resolution by use of the icosahedral symmetry. This procedure gives promise that many other virus structures also can be determined without the use of the isomorphous replacement technique. Although the organization of the major capsid proteins VP1, VP2, and VP3 of Mengo virus is essentially the same as in rhino- and polioviruses, large insertions and deletions, mostly in VP1, radically alter the surface features. In particular, the putative receptor binding "canyon" of human rhinovirus 14 becomes a deep "pit" in Mengo virus because of polypeptide insertions in VP1 that fill part of the canyon. The minor capsid peptide, VP4, is completely internal in Mengo virus, but its association with the other capsid proteins is substantially different from that in rhino- or poliovirus. However, its carboxyl terminus is located at a position similar to that in human rhinovirus 14 and poliovirus, suggesting the same autocatalytic cleavage of VP0 to VP4 and VP2 takes place during assembly in all these picornaviruses.     

Architecture of mammalian fatty acid synthase at 4.5 A resolution

The homodimeric mammalian fatty acid synthase is one of the most complex cellular multienzymes, in that each 270-kilodalton polypeptide chain carries all seven functional domains required for fatty acid synthesis. We have calculated a 4.5 angstrom-resolution x-ray crystallographic map of porcine fatty acid synthase, highly homologous to the human multienzyme, and placed homologous template structures of all individual catalytic domains responsible for the cyclic elongation of fatty acid chains into the electron density. The positioning of domains reveals the complex architecture of the multienzyme forming an intertwined dimer with two lateral semicircular reaction chambers, each containing a full set of catalytic domains required for fatty acid elongation. Large distances between active sites and conformational differences between the reaction chambers demonstrate that mobility of the acyl carrier protein and general flexibility of the multienzyme must accompany handover of the reaction intermediates during the reaction cycle.     

Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution

The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques. No conformational changes can be observed in the tertiary structure of lysozyme compared with that determined in native crystalline forms. The quaternary structure of Fab is that of an extended conformation. The antibody combining site is a rather flat surface with protuberances and depressions formed by its amino acid side chains. The antigen-antibody interface is tightly packed, with 16 lysozyme and 17 antibody residues making close contacts. The antigen contacting residues belong to two stretches of the lysozyme polypeptide chain: residues 18 to 27 and 116 to 129. All the complementarity-determining regions and two residues outside hypervariable positions of the antibody make contact with the antigen. Most of these contacts (10 residues out of 17) are made by the heavy chain, and in particular by its third complementarity-determining region. Antigen variability and antibody specificity and affinity are discussed on the basis of the determined structure.     

The complete atomic structure of the large ribosomal subunit at 2.4 A resolution

The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior.     

Protein-RNA interactions in an icosahedral virus at 3.0 A resolution

Nearly 20 percent of the packaged RNA in bean-pod mottle virus (BPMV) binds to the capsid interior in a symmetric fashion and is clearly visible in the electron density map. The RNA displaying icosahedral symmetry is single-stranded with well-defined polarity and stereochemical properties. Interactions with protein are dominated by nonbonding forces with few specific contacts. The tertiary and quaternary structures of the BPMV capsid proteins are similar to those observed in animal picornaviruses, supporting the close relation between plant comoviruses and animal picornaviruses established by previous biological studies.     

Structure of a thiol monolayer-protected gold nanoparticle at 1.1 A resolution

Structural information on nanometer-sized gold particles has been limited, due in part to the problem of preparing homogeneous material. Here we report the crystallization and x-ray structure determination of a p-mercaptobenzoic acid (p-MBA)-protected gold nanoparticle, which comprises 102 gold atoms and 44 p-MBAs. The central gold atoms are packed in a Marks decahedron, surrounded by additional layers of gold atoms in unanticipated geometries. The p-MBAs interact not only with the gold but also with one another, forming a rigid surface layer. The particles are chiral, with the two enantiomers alternating in the crystal lattice. The discrete nature of the particle may be explained by the closing of a 58-electron shell.     

Architecture of a fungal fatty acid synthase at 5 A resolution

All steps of fatty acid synthesis in fungi are catalyzed by the fatty acid synthase, which forms a 2.6-megadalton alpha6beta6 complex. We have determined the molecular architecture of this multienzyme by fitting the structures of homologous enzymes that catalyze the individual steps of the reaction pathway into a 5 angstrom x-ray crystallographic electron density map. The huge assembly contains two separated reaction chambers, each equipped with three sets of active sites separated by distances up to approximately 130 angstroms, across which acyl carrier protein shuttles substrates during the reaction cycle. Regions of the electron density arising from well-defined structural features outside the catalytic domains separate the two reaction chambers and serve as a matrix in which domains carrying the various active sites are embedded. The structure rationalizes the compartmentalization of fatty acid synthesis, and the spatial arrangement of the active sites has specific implications for our understanding of the reaction cycle mechanism and of the architecture of multienzymes in general.     

Structure of the lambda complex at 2.5 A resolution: details of the repressor-operator interactions

The crystal structure of a complex containing the DNA-binding domain of lambda repressor and a lambda operator site was determined at 2.5 A resolution and refined to a crystallographic R factor of 24.2 percent. The complex is stabilized by an extensive network of hydrogen bonds between the protein and the sugar-phosphate backbone. Several side chains form hydrogen bonds with sites in the major groove, and hydrophobic contacts also contribute to the specificity of binding. The overall arrangement of the complex is quite similar to that predicted from earlier modeling studies, which fit the protein dimer against linear B-form DNA. However, the cocrystal structure reveals important side chain-side chain interactions that were not predicted from the modeling or from previous genetic and biochemical studies.     

Nitrogenase MoFe-protein at 1.16 A resolution: a central ligand in the FeMo-cofactor

A high-resolution crystallographic analysis of the nitrogenase MoFe-protein reveals a previously unrecognized ligand coordinated to six iron atoms in the center of the catalytically essential FeMo-cofactor. The electron density for this ligand is masked in structures with resolutions lower than 1.55 angstroms, owing to Fourier series termination ripples from the surrounding iron and sulfur atoms in the cofactor. The central atom completes an approximate tetrahedral coordination for the six iron atoms, instead of the trigonal coordination proposed on the basis of lower resolution structures. The crystallographic refinement at 1.16 angstrom resolution is consistent with this newly detected component being a light element, most plausibly nitrogen. The presence of a nitrogen atom in the cofactor would have important implications for the mechanism of dinitrogen reduction by nitrogenase.     

Crystallographic structure of the octameric histone core of the nucleosome at a resolution of 3.3 A

The structure of the (H2A-H2B-H3-H4)2 histone octamer has been determined by means of x-ray crystallographic techniques at a resolution of 3.3 angstroms. The octamer is a prolate ellipsoid 110 angstroms long and 65 to 70 angstroms in diameter, and its general shape is that of a rugby ball. The size and shape are radically different from those determined in earlier studies. The most striking feature of the histone octamer is its tripartite organization, that is, a central (H3-H4)2 tetramer flanked by two H2A-H2B dimers. The DNA helix, placed around the octamer in a path suggested by the features on the surface of the protein, appears like a spring holding the H2A-H2B dimers at either end of the (H3-H4)2 tetramer.     

Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein

Ribonuclease H digests the RNA strand of duplex RNA.DNA hybrids into oligonucleotides. This activity is indispensable for retroviral infection and is involved in bacterial replication. The ribonuclease H from Escherichia coli is homologous with the retroviral proteins. The crystal structure of the E. coli enzyme reveals a distinctive alpha-beta tertiary fold. Analysis of the molecular model implicates a carboxyl triad in the catalytic mechanism and suggests a likely mode for the binding of RNA.DNA substrates. The structure was determined by the method of multiwavelength anomalous diffraction (MAD) with the use of synchrotron data from a crystal of the recombinant selenomethionyl protein.     

Structure of tobacco mosaic virus at 3.6 A resolution: implications for assembly

X-ray fiber diffraction analysis of tobacco mosaic virus (TMV) has led to the building of a molecular model of the intact virus, based on a map at 3.6 A resolution derived from five separated Bessel orders. This has been made possible by advances in the solution of the fiber diffraction phase problem. It is now possible to understand much of the chemical basis of TMV assembly, particularly in terms of intersubunit electrostatic interactions and RNA binding. Consideration of the molecular structure in conjunction with physical chemical studies by several groups of investigators suggests that the nucleating aggregate for initiation of TMV assembly is a short (about two turns) helix of protein subunits, probably inhibited from further polymerization in the absence of RNA by the disordering of peptide loop near the inner surface of the virus.