Crystal structure of an initiation factor bound to the 30S ribosomal subunit

Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.     

The crystal structure of TAL effector PthXo1 bound to its DNA target

DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand. Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.     

Crystal engineering: from structure to function

Modern crystal engineering has emerged as a rich discipline whose success requires an iterative process of synthesis, crystallography, crystal structure analysis, and computational methods. By focusing on the molecular recognition events during nucleation and growth, chemists have uncovered new ways of controlling the internal structure and symmetry of crystals and of producing materials with useful chemical and physical properties.     

Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells

Cultured bronchial epithelial and fibroblastic cells from humans were used to study DNA damage and toxicity caused by formaldehyde. Formaldehyde caused the formation of cross-links between DNA and proteins, caused single-strand breaks in DNA, and inhibited the resealing of single-strand breaks produced by ionizing radiation. Formaldehyde also inhibited the unscheduled DNA synthesis that occurs after exposure of cells to ultraviolet irradiation or to benzo[a]pyrene diolexpoxide but at doses substantially higher than those required to inhibit the resealing of x-ray-induced single-strand breaks. Therefore, formaldehyde could exert its mutagenic and carcinogenic effects by both damaging DNA and inhibiting DNA repair.     

Crystal and molecular structure of a thymine phototrimer

Thymine trimer was isolated from a frozen aqueous solution of thymine which was irradiated with ultraviolet light and was presumably formed through the rearrangement of an initial oxetane photoproduct. X-ray diffraction analysis of a single crystal has confirmed the trimeric diol structure and has established the stereoconfiguration of the molecule. The possible importance of the diol structure in photobiology is pointed out.     

Crystal and molecular structure of a pentagonal dodecahedrane

The successful synthesis of a pentagonal dodecahedrane molecule has been achieved. Obtained by a sequence of 20 stereochemically controlled steps beginning with the readily available cyclopentadienide anion, the 1,16-dimethyl derivative has D(3d) symmetry and unusual physical properties. The x-ray crystal structure shows that the alkyl groups cause only small distortions from pure dodecahedral symmetry.     

Crystal and molecular structure of a thymine-thymine adduct

Thymine-thymine adduct is a product isolated from thymine irradiated with ultraviolet light in frozen aqueous solution. This compound is presumably formed through the rearrangement of an initial photoproduct. Single crystal x-ray diffraction analysis has confirmed the molecular formula of the adduct, 5-hydroxy-6-4'-(5'-methylpyrimid-2'-one)-dihydrothymine, except for the possibility of a hydrogen atom on the 3' nitrogren rather than the 1' nitrogen, and has established the stereoconfiguration of the molecule.     

Crystal structure of a lipid G protein-coupled receptor

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P(1)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P(1), resulting in the modulation of immune and stromal cell responses.     

Crystal structure of human ZAG, a fat-depleting factor related to MHC molecules

Zn-alpha2-glycoprotein (ZAG) is a soluble protein that is present in serum and other body fluids. ZAG stimulates lipid degradation in adipocytes and causes the extensive fat losses associated with some advanced cancers. The 2.8 angstrom crystal structure of ZAG resembles a class I major histocompatibility complex (MHC) heavy chain, but ZAG does not bind the class I light chain beta2-microglobulin. The ZAG structure includes a large groove analogous to class I MHC peptide binding grooves. Instead of a peptide, the ZAG groove contains a nonpeptidic compound that may be implicated in lipid catabolism under normal or pathological conditions.     

Crystal structure of serotonin picrate, a donor-acceptor complex

The crystal structure of the red picric acid salt of serotonin was determined by x-ray diffraction methods. The structure consists of parallel hydroxyindole and picrate planes which are intimately stacked with an interplanar separation of 3.3 to 3.4 angstroms. The stacking interaction appears to be of the donor-acceptor (charge-transfer) type, involving specific contacts between picrate nitro groups and atoms of the hydroxyindole moieties. Similar interactions might mediate biological processes involving serotonin.     

Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone

Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.     

Crystal structure of the maltose transporter in a pretranslocation intermediate state

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters convert chemical energy from ATP hydrolysis to mechanical work for substrate translocation. They function by alternating between two states, exposing the substrate-binding site to either side of the membrane. A key question that remains to be addressed is how substrates initiate the transport cycle. Using x-ray crystallography, we have captured the maltose transporter in an intermediate step between the inward- and outward-facing states. We show that interactions with substrate-loaded maltose-binding protein in the periplasm induce a partial closure of the MalK dimer in the cytoplasm. ATP binding to this conformation then promotes progression to the outward-facing state. These results, interpreted in light of biochemical and functional studies, provide a structural basis to understand allosteric communication in ABC transporters.     

Crystal structure of a silyl cation with no coordination to anion and distant coordination to solvent

The crystal structure of a stable silyl cation, triethylsilylium, in the form of its tetrakis (pentafluorophenyl)borate salt [Et(3)Si(+) (C(6)F(5))(4)B(-)] (Et, ethyl) shows no coordination between cation and anion. The closest silicon-fluorine distance is greater than 4 angstroms. A toluene solvent molecule is close enough to cause some deviations from planarity at the silicon, but the silicon-toluene distance is well beyond the sum of the silicon and carbon covalent radii. The toluene molecule is essentially planar and undistorted, as expected if little or no positive charge has been transferred from silicon to toluene.     

The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist

The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.     

Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue

The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.     

Crystal structure of bee-venom phospholipase A2 in a complex with a transition-state analogue

The 2.0 angstroms crystal structure of a complex containing bee-venom phospholipase A2 (PLA2) and a phosphonate transition-state analogue was solved by multiple isomorphous replacement. The electron-density map is sufficiently detailed to visualize the proximal sugars of the enzyme's N-linked carbohydrate and a single molecule of the transition-state analogue bound ot its active center. Although bee-venom PLA2 does not belong to the large homologous Class I/II family that encompasses most other well-studied PLA2s, there is segmental sequence similarity and conservation of many functional substructures. Comparison of the bee-venom enzyme with other phospholipase structures provides compelling evidence for a common catalytic mechanism.     

基因组原位杂交中封阻DNA制备方法研究

在基因组原位杂交中,适当的封阻可以大大提高基因组原位杂交的效率。本研究采用煮沸法、超声波剪切法对大白菜基因组DNA进行剪切,研究了大白菜封阻DNA的制备方法。结果表明:珠沸法效率高,操作简单,当煮沸70 min DNA片段大小主要集中在200～500 bp,适于在基因组原位杂交中作为封阻。研究结果为基因组原位杂交的应用奠定了基础。