1. Femtosecond time resolution in x-ray diffraction experiments - Neutze, Richard; Hajdu, Janos
This paper presents the theoretical background for a synthesis of femtosecond spectroscopy and x-ray diffraction. When a diffraction quality crystal with 0.1–0.3 mm overall dimensions is photoactivated by a femtosecond laser pulse (physical length = 0.3 ?m), the evolution of molecules at separated points in the crystal will not be simultaneous because a finite time is required for the laser pulse to propagate through the body of the crystal. Utilizing this lack of global crystal synchronization, topographic x-ray diffraction may enable femtosecond temporal resolution to be achieved from reflection profiles in the diffraction pattern with x-ray exposures of picosecond or...

2. X-ray emission from clusters and groups of?galaxies - Mushotzky, Richard
Recent major advances in x-ray imaging and spectroscopy of clusters have allowed the determination of their mass and mass profile out to ?1/2 the virial radius. In rich clusters, most of the baryonic mass is in the gas phase, and the ratio of mass in gas/stars varies by a factor of 2–4. The baryonic fractions vary by a factor of ?3 from cluster to cluster and almost always exceed 0.09 h50?[3/2] and thus are in fundamental conflict with the assumption of ? = 1 and the results of big bang nucleosynthesis. The derived Fe abundances are 0.2–0.45 solar, and the...

3. X-ray-induced mutations in mouse embryonic stem?cells - Thomas, James W.; LaMantia, Christian; Magnuson, Terry
Deletion complexes consisting of multiple chromosomal deletions induced at single loci can provide a means for functional analysis of regions spanning several centimorgans in model genetic systems. A strategy to identify and map deletions at any cloned locus in the mouse is described here. First, a highly polymorphic, germ-line competent F1(129/Sv-+Tyr+p × CAST/Ei) mouse embryonic stem cell line was established. Then, x-ray and UV-induced mutagenesis was performed to determine the feasibility of generating deletion complexes throughout the mouse genome. Reported here are the selection protocols, induced mutation frequencies, cytogenetic and extensive molecular analysis of mutations at the X-chromosome-linked hypoxanthine phosphoribosyltransferase...

4. X-ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine. - Lipscomb, L A; Peek, M E; Morningstar, M L; Verghis, S M; Miller, E M; Rich, A; Essigmann, J M; Williams, L D
We have determined the x-ray structure of a DNA fragment containing 7,8-dihydro-8-oxoguanine (G(O)). The structure of the duplex form of d(CCAGOCGCTGG) has been determined to 1.6-A resolution. The results demonstrate that GO forms Watson-Crick base pairs with the opposite C and that G(O) is in the anti conformation. Structural perturbations induced by C.G(O)anti base pairs are subtle. The structure allows us to identify probable elements by which the DNA repair protein MutM recognizes its substrates. Hydrogen bond donors/acceptors within the major groove are the most likely element. In that groove, the pattern of hydrogen-bond donors/acceptors of C.G(O)anti is unique. Additional...

5. X-ray structure of sensory rhodopsin II at 2.1-? resolution - Royant, Antoine; Nollert, Peter; Edman, Karl; Neutze, Richard; Landau, Ehud M.; Pebay-Peyroula, Eva; Navarro, Javier
Sensory rhodopsins (SRs) belong to a subfamily of heptahelical transmembrane proteins containing a retinal chromophore. These photoreceptors mediate the cascade of vision in animal eyes and phototaxis in archaebacteria and unicellular flagellated algae. Signal transduction by these photoreceptors occurs by means of transducer proteins. The two archaebacterial sensory rhodopsins SRI and SRII are coupled to the membrane-bound HtrI and HtrII transducer proteins. Activation of these proteins initiates phosphorylation cascades that modulate the flagellar motors, resulting in either attractant (SRI) or repellent (SRII) phototaxis. In addition, transducer-free SRI and SRII were shown to operate as proton pumps, analogous to bacteriorhodopsin. Here,...

6. Ultra-small-angle x-ray-scattering study: preliminary experiments in colloidal suspensions. - Matsuoka, H; Kakigami, K; Ise, N; Kobayashi, Y; Machitani, Y; Kikuchi, T; Kato, T
We report here a multiple-reflection type ultra-small-angle x-ray-scattering apparatus constructed according to the principle proposed by Bonse and Hart. The ultra-small-angle x-ray scattering technique enables us to investigate the distribution of solutes or particles in turbid systems, which could not be studied by the conventional light-scattering and microscopic methods. By this method, the density fluctuation from an order of several thousand angstroms to several micrometers is now detectable. In this paper, the ultra-small-angle x-ray scattering apparatus was calibrated by using well-defined latex particles.

7. Soft x-ray magnetic circular dichroism: a probe for studying paramagnetic bioinorganic systems. - van Elp, J; George, S J; Chen, J; Peng, G; Chen, C T; Tjeng, L H; Meigs, G; Lin, H J; Zhou, Z H; Adams, M W
Soft x-ray magnetic circular dichroism was used to study a paramagnetic bioinogranic system. We measured the Fe L edges of Pyrococcus furiosus rubredoxin, using circularly polarized synchrotron radiation, a split-coil super-conducting magnet, low sample temperatures, and fluorescence detection. The observed dichroism effect is strong (30%) and in general agreement with the calculation. The method is element- and oxidation state-specific, and the data can be interpreted by established theoretical procedures. Soft x-ray magnetic circular dichroism demonstrates enormous potential as a probe for studying paramagnetic systems in biology, chemistry, and material science.

8. Protein folding at the air–water interface studied with x-ray reflectivity - Gidalevitz, David; Huang, Zhengqing; Rice, Stuart A.
We report the results of x-ray reflectivity measurements of thin films formed by different water-soluble proteins at the air–aqueous solution interface. It is demonstrated that glucose oxidase, alcohol dehydrogenase, and urease molecules denaturate at the air–aqueous solution interface to form 8- to 14-?-thick peptide sheets. X-ray reflectivity data indicate that the spreading of a lipid monolayer at the aqueous solution surface before protein injection does not prevent proteins from unfolding. On the other hand, crosslinking of proteins results in intact enzyme layers at the subphase surface. A model that involves interaction of glucose oxidase molecules with a phospholipid monolayer is...

9. X-ray diffraction study of the binding of the antisickling agent 12C79 to human hemoglobin. - Wireko, F C; Abraham, D J
The hemoglobin binding site of the antisickling agent 12C79 has been determined by x-ray crystallography. 12C79 is recognized as one of the first molecules to reach clinical trials that was designed, de novo, from x-ray-determined atomic coordinates of a protein. Several previous attempts to verify the proposed Hb binding sites via crystallographic studies have failed. Using revised experimental procedures, we obtained 12C79-deoxyhemoglobin crystals grown after reaction with oxyhemoglobin and cyanoborohydride reduction to stabilize the Schiff base linkage. The difference electron-density Fourier maps show that two 12C79 molecules bind covalently to both symmetry-related N-terminal amino groups of the hemoglobin alpha chains....

10. Intracellular structures of normal and aberrant Plasmodium falciparum malaria parasites imaged by soft x-ray?microscopy - Magowan, Cathleen; Brown, John T.; Liang, Joy; Heck, John; Coppel, Ross L.; Mohandas, Narla; Meyer-Ilse, Werner
Soft x-ray microscopy is a novel approach for investigation of intracellular organisms and subcellular structures with high spatial resolution. We used x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes and in infected erythrocytes treated with cysteine protease inhibitors. Investigations in normal red blood cells enabled us to recognize anomalies in parasite structures resulting from growth under unfavorable conditions. X-ray microscopy facilitated detection of newly elaborated structures in the cytosol of fixed, unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. In cysteine protease inhibitor-treated, infected erythrocytes,...

11. X-ray scattering indicates that the leucine zipper is a coiled coil. - Rasmussen, R; Benvegnu, D; O'Shea, E K; Kim, P S; Alber, T
Dimerization of the bZIP class of eukaryotic transcriptional control proteins requires a sequence motif called the leucine zipper. We have grown two distinct crystal forms of a 33-amino acid peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4. This peptide is known to form a dimer of parallel helices in solution. X-ray scattering from both crystal forms shows reflections that are diagnostic of coiled coils. The most notable reflections occur at approximately 5.2 A resolution and correspond to the pitch of helices in coiled coils. There is no diffraction maximum near 5.4 A, the characteristic pitch of...

12. Disordered water within a hydrophobic protein cavity visualized by x-ray crystallography - Yu, B.; Blaber, M.; Gronenborn, A. M.; Clore, G. M.; Caspar, D. L. D.
Water in the hydrophobic cavity of human interleukin 1?, which was detected by NMR spectroscopy but was invisible by high resolution x-ray crystallography, has been mapped quantitatively by measurement and phasing of all of the low resolution x-ray diffraction data from a single crystal. Phases for the low resolution data were refined by iterative density modification of an initial flat solvent model outside the envelope of the atomic model. The refinement was restrained by the condition that the map of the difference between the electron density distribution in the full unit cell and that of the atomic model be flat...

13. Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction. - Kamikubo, H; Kataoka, M; Váró, G; Oka, T; Tokunaga, F; Needleman, R; Lanyi, J K
X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5 degrees Celsius. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The...

14. Determination of the absolute configuration of (+)-neopentyl-1-d alcohol by neutron and x-ray diffraction analysis. - Yuan, H S; Stevens, R C; Bau, R; Mosher, H S; Koetzle, T F
The absolute configuration of (+)-neopentyl-1-d alcohol, prepared by the reduction of 2,2-dimethylpropanal-1-d by actively fermenting yeast, has been determined to be S by neutron diffraction. The neutron study was carried out on the phthalate half ester of neopentyl-1-d alcohol, crystallized as its strychnine salt. The absolute configuration of the (-)-strychninium cation was first determined by an x-ray anomalous dispersion study of its iodide salt. The chiral skeleton of strychnine then served as a reference from which the absolute configuration of the -O-CHD-C(CH3)3 group of neopentyl phthalate was determined. Difference Fourier maps calculated from the neutron data showed unambiguously that the...

15. Isolation of x-ray-inducible transcripts from radioresistant human melanoma cells. - Boothman, D A; Meyers, M; Fukunaga, N; Lee, S W
Twelve x-ray-induced transcripts (xips), differentially expressed 8- to 230-fold in x-irradiated versus unirradiated radioresistant human melanoma (U1-Mel) cells, were isolated as cDNA clones (xip1 through xip12) after four rounds of differential hybridization. Northern analyses revealed rare, medium, and abundant xips, ranging in size from 1.2 to 10 kb. All transcripts were transiently expressed and induced by low, but not by high (> 600 cGy), doses of radiation. Three transcripts (xip4, -7, and -12) were induced only by ionizing radiation, and many (i.e., xip1, -2, -3, -5, -6, -8, -9, -10, and -11) were also induced by UV irradiation or phorbol...

16. Observation of the light-triggered binding of pyrone to chymotrypsin by Laue x-ray crystallography. - Stoddard, B L; Koenigs, P; Porter, N; Petratos, K; Petsko, G A; Ringe, D
Crystals of gamma-chymotrypsin inhibited with the photodissociable group trans-p-diethylamino-o-hydroxy-alpha-methylcinnamate were irradiated with a 1-msec flash from a high-energy xenon flashlamp in the presence of the mechanism-based inhibitor 3-benzyl-6-chloro-2-pyrone. The ensuing reaction was monitored by collection of sequential, single-exposure Laue x-ray diffraction patterns. The experiment was also performed in solution to verify the regeneration of catalytic activity and the subsequent inhibition of the enzyme by pyrone after photolysis. The resulting crystallographic structures show the presence of covalently bound cinnamate prior to photolysis, the generation of "free" enzyme after irradiation of the crystal, and the slow formation of a pyrone-inhibited complex several...

17. Interference fine structure and sarcomere length dependence of the axial x-ray pattern from active single muscle fibers - Linari, M.; Piazzesi, G.; Dobbie, I.; Koubassova, N.; Reconditi, M.; Narayanan, T.; Diat, O.; Irving, M.; Lombardi, V.
Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2–3.2 ?m, the M3 x-ray reflection, associated with the repeat of myosin heads along the filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between myosin and actin filaments, showing that it comes from myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere...

18. X-ray crystal structure of an anti-Buckminsterfullerene antibody Fab fragment: Biomolecular recognition of C60 - Braden, Bradford C.; Goldbaum, Fernando A.; Chen, Bi-Xing; Kirschner, Austin N.; Wilson, Stephen R.; Erlanger, Bernard F.
We have prepared a monoclonal Buckminsterfullerene specific antibody and report the sequences of its light and heavy chains. We also show, by x-ray crystallographic analysis of the Fab fragment and by model building, that the fullerene binding site is formed by the interface of the antibody light and heavy chains. Shape-complementary clustering of hydrophobic amino acids, several of which participate in putative stacking interactions with fullerene, form the binding site. Moreover, an induced fit mechanism appears to participate in the fullerene binding process. Affinity of the antibody–fullerene complex is 22 nM as measured by competitive binding. These findings should be...

19. Protein hydration in solution: Experimental observation by x-ray and neutron scattering - Svergun, D. I.; Richard, S.; Koch, M. H. J.; Sayers, Z.; Kuprin, S.; Zaccai, G.
The structure of the protein–solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solutions. The analysis of the protein–solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density ?10% larger than that of the bulk solvent in the...

20. Three-dimensional diffuse x-ray scattering from crystals of?Staphylococcal?nuclease - Wall, Michael E.; Ealick, Steven E.; Gruner, Sol M.
We have developed methods for obtaining and characterizing three-dimensional maps of the reciprocal-space distribution of diffuse x-ray scattering from protein crystals, and have used the methods to study the nature of disorder in crystals of Staphylococcal nuclease. Experimentally obtained maps are 99.5% complete in the reciprocal-space resolution range of 10 ?–2.5 ?, show symmetry consistent with the P41 space group of the unit cell, and are highly reproducible. Quantitative comparisons of the data with three-dimensional simulations imply liquid-like motions of the protein [Caspar, D.?L.?D., Clarage, J., Salunke, D.?M. & Clarage, M. (1988) Nature (London) 332, 659–662], with a correlation length...

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