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Single crystals of the RuvC protein, an Escherichia coli endonuclease specific for Holliday junctions, were grown by the microdialysis method. The crystals belong to the space group P 2 1 , with unit cell dimensions a = 72·8 A, b = 139·6 A, c = 32·4 A and β = 93·0°, and contain four molecules in an asymmetric unit. Diffraction data to a Bragg spacing of 2·5 A resolution has been obtained using a synchrotron X-ray source.

The vitelline membrane outer layer protein I (VMO-I), which is isolated from the vitelline membrane outer layer of hen's eggs, has been crystallized from an acetate buffer solution by the hanging-drop method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 62.42 A, b = 110.52 A, c = 44.15 A. There are two molecules (M(r) = 18,000) per asymmetric unit. The crystals diffract to at least 2.2 A Bragg spacings.

Crystals as large as 0.5 X 0.3 X 0.2 mm of purified arginine-transfer RNA from Escherichia coli have been prepared by a vapour diffusion method. X-ray diffraction photographs showed that the crystals gave reflections up to 3.7 A spacing. They have a trigonal space group P31 2 1 (OR P32 2 1) and cell-dimensions a=97.2, b=97.2, c=94.8A. Crystals of a mercury derivative of this transfer RNA have also been obtained, and an X-ray diffusion photography of one of them is presented. Formylmethionine-transfer RNA from E. coli was crystallized in various forms, and the appearance of the polymorphs was found to depend upon the amount of spermine in the solution from which the crystallization took place. Crystals of tyrosine-transfer RNA and glycine-transfer RNA have also been obtained.

Electron microscopy was applied to thin crystals of yeast tRNAPhe. The crystals embedded in glucose yield Bragg reflections with a spacing smaller than 4 . The measurement of radiation damage rate demonstrates that they are 4 to 14 times less susceptible to electron exposures than protein crystals embedded in glucose.

The site-directed mutagenesis opens the doors to investigations of the fundamental nature of proteins and to additions of new valuable characters to proteins. In such circumstances, it is desired to speed up the X-ray structure analyses of protein molecules. Area detectors are expected as one of the promising outcomes of recent technical developments and it can collect diffraction intensities many times more rapidly than current instruments. One can design a new protein from the many results of these protein structure determinations and also of the closely related protein structures. [J. Cryst. Soc. Jpn. 29, 27 (1987) ] .

A method was developed to visualize individual DNA molecules in solution under a fluorescent microscope connected to a highly sensitive video camera. DNA stained with a fluorescent dye, DAPI, revealed thin extended filaments, thicker filaments and rapidly transforming folded structures dependent upon the solution conditions. Structural transitions were observed and recorded as video images. Our observations indicated the possibility that DNA has the ability of supercoiling itself. This DAPI staining method will have wide possible application in the study of DNA and chromatin.

Infrared and Raman spectra of aqueous poly(rA-rU)·poly(rA-rU), the double-helical complex containing strands of alternating riboadenylate and ribouridylate residues, display significant differences from one another and from corresponding spectra of poly(rA)·poly(rU), the double-helical complex of riboadenylate and ribouridylate homopolymers. Parallel studies on the copolymer and homopolymer complexes by cesium sulfate density gradient centrifugation, ultraviolet absorption spectroscopy, hydrogenion titration, 1-N oxidation of adenine residues by monoperphthalic acid and X-ray diffraction reveal, however, that the geometry of base pairing between adenine and uracil is closely similar in each complex and apparently of the Watson-Crick type. Therefore the differences observed between vibrational spectra of poly (rA-rU)·poly (rA-rU) and poly(rA)·poly(rU) are not due to different base-pairing schemes but may be attributed to differences in vibrational coupling between vertically stacked bases. Vibrational coupling may also account for the differences between infrared and Raman spectra of the same complex. Thus, the present results indicate that infrared and Raman frequencies of RNA in the region 1750–1550 cm−1 should be dependent on the base sequence.

Four fragments (named K, L, M, and N) of Escherichia coli formylmethionine transfer RNA have been prepared by a partial digestion with ribonuclease T1 followed by a chromatographic separation with a DEAE–Sphadex (A-25) column and then a DEAE–cellulose column. The fragment K is the anticodon fragment with 19 nucleotides (previously reported). L is a fragment with 57 nucleotides involving the 3′-terminal (CCA). M is a fragment with 51 nucleotides which is equal to L except that M lacks 6 nucleotides at the 3′-terminal. N is a fragment with 20 nucleotides which involved the 5′-terminal and corresponds to the complementary half to L. The infrared absorption spectrum has been observed of each of these fragments and two equimolar mixtures L + N and M + N in D2O solutions at several temperatures. The results indicate that at 37°C, K has about 4 hydrogen-bonded base-pairs, L about 11, and M about 13. On the other hand, fragment N is found to have only three weak G-C pairs. For both L + N and M + N, 15–17 strong base pairs are found. The observations give direct support to the clover-leaf structure and at the same time provide information on the stability of each of the four arms in the structure.

Ultraviolet absorbance versus temperature profile has been examined of seven helical nucleic acids in aqueous solutions with and without poly-L-ornithine. The hyperchromicity observed at the melting temperature of each nucleic acid is found to be lowered linearly as the amount of poly-L-ornithine added to the solution. This fact is taken as indicating that there is a stoichiometric complex of polyornithine plus nucleic acid formed. The ornithine/nucleotide mole ratio was determined to be 1/1 for the complexes of calf thymus DNA, 3) poly dAT, rice dwarf virus RNA, and poly rAU. The ratio was found to be 2/3 for the complexes of poly (A+U) and poly (A+2U), and 1/2 for the complex of poly (I+C).

15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) activates a nuclear receptor heterodimer, peroxisome proliferators-activated receptor gamma (PPARgamma)/ retinoid X receptor (RXRalpha) through covalent binding to Cys285 in PPARgamma ligand-binding domain (LBD). Here, we present the 1.9A crystal structure of C285S mutant LBD complexed with 15d-PGJ(2), corresponding to the non-covalently bound state. The ligand lies adjacent to a hydrogen-bond network around the helix H2 and the nearby beta-sheet. Comparisons with previous structures clarified the relationships between PPARgamma function and conformational alterations of LBD during the process of covalently binding ligands, such as 15d-PGJ(2), and thus suggested a mechanism, by which these ligands modulate PPARgamma/RXRalpha function through conformational changes of the loop following helix H2' and the beta-sheet.

Background: Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, α and β , which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. Results: Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, α and β , are jointly folded into an ellipsoidal, single domain structure belonging to the α / β -sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split β α β motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. Conclusions: The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range. The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.

Two enzymes, dUTP pyrophosphatase and uracil-DNA glycosylase, prevent the misincorporation of uracil into the genome in distinct manners. The atomic structures of these proteins complexed with substrate analogs reveal the structural basis for uracil recognition and suggest a novel mechanism of DNA repair.

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.

Background: Homologous recombination is crucial for genetic diversity and repairing damaged chromosomes. In Escherichia coli cells, the RuvA, RuvB and RuvC proteins participate in the processing of an important intermediate, the Holliday junction. The RuvA–RuvB protein complex facilitates branch migration of the junction, depending on ATP hydrolysis. The atomic structure of RuvA should enable critical questions to be addressed about its specific interactions with the Holliday junction and the RuvB protein. Results: The crystal structure of RuvA shows the tetrameric molecules with a fourfold axis at the center. Each subunit consists of three distinct domains, some of which contain important secondary structure elements for DNA binding. Together with the detailed structural information, the biochemical assays of various mutant RuvA proteins and domains, isolated by partial proteolysis, allowed us to define the functional roles of these domains in Holliday junction binding and the RuvB interaction. Conclusions: The RuvA molecule is formed by four identical subunits, each with three domains, I, II and III. The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction. Domain III is weakly attached to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III. Functional analyses of the mutant proteins and the partial digestion products, including Holliday junction binding and branch-migration assays, revealed that domain III and the preceding loop are crucial for RuvB binding and branch migration, although this region is not required for the junction–DNA binding.

SummaryThe multiprotein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold-containing CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the “histone code” beyond canonical nucleosome proteins.

Summary DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus . The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae ; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60° inclination at the central thymine dimer. The adenine base complementary to the 5′ side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.

Bacteriophage T4 endonuclease V, which is an excision-repair enzyme specific to pyrimidine dimers within DNA, has been crystallized from polyethylene glycol 4000 solution by a vapour diffusion technique. The unit cell is monoclinic, space group P21 with unit cell parameters: a = 41.4 A , b = 40.1 A , c = 37.5 A , β = 90.01° . The unit cell contains two 16,000 Mr molecules. The crystals diffract X-rays beyond 2.3 A resolution and are suitable for structural analysis at high resolution.

We have determined the complete nucleotide sequence of a 5.3-kb long genomic DNA fragment of the fission yeast Schizosaccharomyces pombe that encodes DNA topoisomerase II. It contains a 4293 bp long single open reading frame. The predicted polypeptide has 1431 residues (mol. wt 162,000) and shows three characteristic domains; the large C-terminal region, which consists of alternating acidic-basic stretches and might be a chromatin-binding domain, the NH2 half domain homologous to the ATP-binding gyrB subunit of bacterial gyrase and the central-to-latter part which is homologous to the NH2 domain of the catalytic gyrA subunit, suggesting a possible evolutionary consequence of the gene fusion of the bacterial gyrase subunits into the eucaryotic DNA topoisomerase II gene. We have found that the cloned fission yeast TOP2 gene can complement the budding yeast top2 mutation, although the fission yeast TOP2 protein sequence is only 50% homologous to the recently determined sequence of budding yeast (J.C. Wang, personal communication). Conversely, the budding yeast TOP2 gene can complement the fission yeast top2 mutations, indicating that their DNA topoisomerase II genes are functionally exchangeable.

Individual DNA molecules in solution can be visualized under a fluorescence microscope by using the DNA-binding dye 4′,6-diamidino-2-phenylindole and can be recorded on video as mobile structures (Morikawa & Yanagida, 1981). DNA in the presence of 10mm-MgCl2 was found to adhere to the glass surface, so that 4′,6-diamidino-2-phenylindole-stained DNA can be filmed as still images. Fluorescence micrographs of DNA (bacteriophages T4, T3 and λ, yeast and chicken erythrocyte) taken by the present procedure are better in resolution than those obtained by video, showing structural details of DNA molecules hitherto not observed in solution. In the specimens prepared at the reduced shear stress, the folded particles and the short thick filaments were abundant. The shear stream extended them into the wavy and the straight thin filaments. The lengths of the thin filaments seen in viral DNA correlated well with those determined by electron microscopy. Our results suggest that a DNA molecule in solution forms a certain kind of supercoil of its own accord.

To facilitate the preparation of ribonuclease H from Escherichia coli in an amount sufficient for crystallographic studies, we have constructed an overproduction system for the enzyme. The structural gene for the enzyme was subcloned from pSK750 (Kanaya, S., and Crouch, R. J. (1983) J. Biol. Chem. 258, 1276-1281) to make a plasmid vector pPL801, in which the gene was under the control of bacteriophage lambda PL promoter. Thermal induction of the gene accumulated the enzyme in E. coli N4830-1 to approximately 8% of the total cytosolic protein. The level of production of the enzyme in N4830-1 harboring pPL801 was 14 mg/liter culture, which was 3000 times as high as that in the host cell. The enzyme was purified with a yield of more than 80% and crystallized by utilizing the property that the solubility of the enzyme decreased at pH values close to its isoelectric point (pI = 9). Crystals were grown by successive seeding (hanging drop method) for x-ray crystallographic analysis. The crystals belong to space group P212121 with unit cell dimensions of alpha = 44.1 A, b = 87.0 A, c = 35.5 A and contain one molecule in an asymmetric unit. They diffracted x-rays beyond 2.5 A resolution.

Several types of crystals were grown from 5S rRNA, which was purified from the highly thermophilic bacterium, Thermus thermophilus HB8. Crystal lattice parameters were determined by X-ray and electron diffraction. One type of crystal was suitable for electron microscopy after staining with uranyl acetate. Two projections derived from micrographs taken at different tilt angles were processed for image analysis. This result enabled us to deduce the arrangement of molecules within the crystal lattice.

Infrared absorption spectrum of an oriented film of deoxyribonucleic acid from Micrococcus lysodeikticus has been examined in its A and B forms. In the A form a weak band was observed at 807 cm−1, while in the B form this is absent and a broad and weak band appears at 832 cm−1.

Publisher Summary This chapter discusses the structure of a thin crystal of tRNA. In a study described in the chapter, crystals of yeast tRNA sufficiently thin to avoid multiple scattering of an electron beam were grown and negatively stained with uranyl acetate, and their images were taken with a JEM-200CX. The crystal form was the same as used in X-ray diffraction. The images of ice-embedded tRNA crystal were taken with a JEM-4000EX equipped with a superfluid He cryo-stage. Two-dimensional spatial filtering of electron microscopic crystalline images was performed. The simulated projection images of tRNA crystal were obtained in the reverse manner of three-dimensional image reconstruction. The original electron micrographs of tRNA thin crystals, negatively stained and ice-embedded in the cryo stage, were compared.

In order to investigate the relationships between the three-dimensional structure and the enzymic activity of E. coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of three functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized. Their three-dimensional structures were determined by x-ray crystallography. Although the entire backbone structures of these mutants were not affected by the replacements, very localized conformational changes were observed around the Mg(2+)-binding site. The substitution of an amide group for a negatively charged carboxyl group in common induces the formation of new hydrogen bond networks, presumably due to the cancellation of repulsive forces between carboxyl side chains with negative charges. These conformational changes can account for the loss of the enzymic activity in the mutants, and suggest a possible role for Mg2+ in the hydrolysis. Since the 3 replaced acidic residues are completely conserved in sequences of reverse transcriptases from retroviruses, including human immunodeficiency virus, the concepts of the catalytic mechanism deduced from this structural analysis can also be applied to RNase H activity in reverse transcriptases.

The metabotropic glutamate receptors (mGluRs) are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. Here we have determined three different crystal structures of the extracellular ligand-binding region of mGluR1--in a complex with glutamate and in two unliganded forms. They all showed disulphide-linked homodimers, whose 'active' and 'resting' conformations are modulated through the dimeric interface by a packed alpha-helical structure. The bi-lobed protomer architectures flexibly change their domain arrangements to form an 'open' or 'closed' conformation. The structures imply that glutamate binding stabilizes both the 'active' dimer and the 'closed' protomer in dynamic equilibrium. Movements of the four domains in the dimer are likely to affect the separation of the transmembrane and intracellular regions, and thereby activate the receptor. This scheme in the initial receptor activation could be applied generally to G-protein-coupled neurotransmitter receptors that possess extracellular ligand-binding sites.

Infrared absorption spectrum of an oriented film of deoxyribonucleic acid from Micrococcus lysodeikticus has been examined in its A and B forms. In the A form a weak band was observed at 807 cm−1, while in the B form this is absent and a broad and weak band appears at 832 cm−1.

WE have observed the infrared spectrum of a fragment of a transfer RNA (tRNA) in aqueous solution and obtained evidence for the existence of the hydrogen-bonded base pairs.

Background In the early stage of eukaryotic DNA replication, the template DNA is unwound by the MCM helicase, which is activated by forming a complex with the Cdc45 and GINS proteins. The eukaryotic GINS forms a heterotetramer, comprising four types of subunits. On the other hand, the archaeal GINS appears to be either a tetramer formed by two types of subunits in a 2:2 ratio (α2β2) or a homotetramer of a single subunit (α4). Due to the low sequence similarity between the archaeal and eukaryotic GINS subunits, the atomic structures of the archaeal GINS complexes are attracting interest for comparisons of their subunit architectures and organization. Results We determined the crystal structure of the α2β2 GINS tetramer from Thermococcus kodakaraensis (TkoGINS), comprising Gins51 and Gins23, and compared it with the reported human GINS structures. The backbone structure of each subunit and the tetrameric assembly are similar to those of human GINS. However, the location of the C-terminal small domain of Gins51 is remarkably different between the archaeal and human GINS structures. In addition, TkoGINS exhibits different subunit contacts from those in human GINS, as a consequence of the different relative locations and orientations between the domains. Based on the GINS crystal structures, we built a homology model of the putative homotetrameric GINS from Thermoplasma acidophilum (TacGINS). Importantly, we propose that a long insertion loop allows the differential positioning of the C-terminal domains and, as a consequence, exclusively leads to the formation of an asymmetric homotetramer rather than a symmetrical one. Conclusions The DNA metabolizing proteins from archaea are similar to those from eukaryotes, and the archaeal multi-subunit complexes are occasionally simplified versions of the eukaryotic ones. The overall similarity in the architectures between the archaeal and eukaryotic GINS complexes suggests that the GINS function, directed through interactions with other protein components, is basically conserved. On the other hand, the different subunit contacts, including the locations and contributions of the C-terminal domains to the tetramer formation, imply the possibility that the archaeal and eukaryotic GINS complexes contribute to DNA unwinding reactions by significantly different mechanisms in terms of the atomic details.